Ocular applications of silk-based products

ABSTRACT

The embodiments disclosed and embraced by the present invention include silk-based products useful in the treatment, diagnosis, palliation, and/or amelioration of ocular diseases or conditions of the eye including those of the structures of the eye and surrounding tissue. Such silk-based products may effect beneficial outcomes alone or in combination with therapeutic modalities, compounds or medicaments.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to 62/584,153 filed on Nov. 10, 2017entitled Manufacture and Uses of Silk Fibroin, 62/659,209 filed Apr. 18,2018 entitled Ocular Silk-Based Products and Methods of Use, and62/680,371 filed Jun. 4, 2018 entitled Ocular Silk-Based Products andMethods of Use, the contents of each of which are herein incorporated byreference in their entirety.

FIELD OF THE INVENTION

The present invention relates to formulations and methods of maintainingand delivering therapeutic agents for ocular indications. Specificallyprovided are silk-based product formulations.

BACKGROUND OF THE INVENTION

Silk is a naturally occurring polymer. Most silk fibers are derived fromsilkworm moth (Bombyx mori) cocoons and include silk fibroin and sericinproteins. Silk fibroin is a fibrous material that forms a polymericmatrix bonded together with sericin. In nature, silk is formed from aconcentrated solution of these proteins that are extruded throughsilkworm spinnerets to produce a highly insoluble fiber. These fibershave been used for centuries to form threads used in garments and othertextiles.

Many properties of silk make it an attractive candidate for productsserving a variety of industries. Polymer strength and flexibility hassupported classical uses of silk in textiles and materials, while silkbiocompatibility has gained attention more recently for applications inmedicine.

Although a variety of products and uses related to silk are beingdeveloped, there remains a need for silk-based products that can meetthe demands of modern medicine. Additionally, there remains a need forsilk-based products that can leverage silk polymer strength,flexibility, and biocompatibility to meet needs in the field ofmedicine. The present disclosure addresses these needs by providingsilk-based products and related methods of preparation and use inmedical applications.

SUMMARY OF THE INVENTION

In some embodiments, the present disclosure provides a silk-basedproduct (SBP) that includes processed silk and an ocular therapeuticagent. The SBP may be in the shape of a rod. The SBP may include fromabout 0.1% to about 98%, weight per weight (w/w), of the oculartherapeutic agent. The SBP may include from about 15% to about 95% (w/w)of the ocular therapeutic agent. The SBP may include from about 5% toabout 85% (w/w) of the ocular therapeutic agent. The SBP may includefrom about 45% to about 75% (w/w) of the ocular therapeutic agent. TheSBP may include a ratio of ocular therapeutic agent concentration toprocessed silk concentration of from about 0.01 to about 4.2. The ratioof ocular therapeutic agent concentration to processed silkconcentration may be from about 0.01 to about 1. The ratio of oculartherapeutic agent concentration to processed silk concentration may befrom about 1 to about 4.2. The SBP may include one or more excipients.The one or more excipients may include one or more of lactose, sorbitol,sucrose, mannitol, lactose USP, Starch 1500, microcrystalline cellulose,Avicel®, phosphate salts, sodium chloride, hydrochloric acid,polysorbate 80, potassium phosphate monobasic, potassium phosphatedibasic, sodium phosphate dibasic, sodium phosphate monobasic, phosphatebuffer, phosphate buffered saline, sodium hydroxide, dibasic calciumphosphate dehydrate, tartaric acid, citric acid, fumaric acid, succinicacid, malic acid, polyvinylpyrrolidone, copolymers of vinylpyrrolidoneand vinylacetate, hydroxypropylcellulose, hydroxyethylcellulose,hydroxypropylmethylcellulose, polyvinyl alcohol, polyethylene glycol,acacia, and sodium carboxymethylcellulose. The SBP may include sucrose.The SBP may include at least one excipient, wherein the at least oneexcipient is present at a concentration of from about 0.01% (w/w) toabout 20% (w/w). The SBP may include a density of from about 0.7 g/mL toabout 1.4 g/mL. Rod-shaped SBPs may have a diameter of from about 0.1 mmto about 1.5 mm. Rod-shaped SBPs may have a length of from about 8 mm toabout 12 mm. The SBP may be a hydrogel. The SBP may be freeze dried. TheSBP may include at least one excipient selected from one or more membersof the group consisting of sorbitol, triethylamine, 2-pyrrolidone,alpha-cyclodextrin, benzyl alcohol, beta-cyclodextrin, dimethylsulfoxide, dimethylacetamide (DMA), dimethylformamide, ethanol,gamma-cyclodextrin, glycerol, glycerol formal, hydroxypropylbeta-cyclodextrin, kolliphor 124, kolliphor 181, kolliphor 188,kolliphor 407, kolliphor EL (cremaphor EL), cremaphor RH 40, cremophorRH 60, dalpha-tocopherol, PEG 1000 succinate, polysorbate 20,polysorbate 80, solutol HS 15, sorbitan monooleate, poloxamer-407,poloxamer-188, Labrafil M-1944CS, Labrafil M-2125CS, Labrasol, Gellucire44/14, Softigen 767, mono- and di-fatty acid esters of PEG 300, PEG 400,or PEG 1750, kolliphor RH60, N-methyl-2-pyrrolidone, castor oil, cornoil, cottonseed oil, olive oil, peanut oil, peppermint oil, saffloweroil, sesame oil, soybean oil, hydrogenated vegetable oils, hydrogenatedsoybean oil, medium chain triglycerides of coconut oil, medium chaintriglycerides of palm seed oil, beeswax, d-alpha-tocopherol, oleic acid,medium-chain mono-glycerides, medium-chain di-glycerides,alpha-cyclodextrin, betacyclodextrin, hydroxypropyl-beta-cyclodextrin,sulfo-butylether-beta-cyclodextrin, hydrogenated soyphosphatidylcholine, distearoylphosphatidylglycerol,L-alphadimyristoylphosphatidylcholine,L-alpha-dimyristoylphosphatidylglycerol, PEG 300, PEG 300caprylic/capric glycerides (Softigen 767), PEG 300 linoleic glycerides(Labrafil M-2125CS), PEG 300 oleic glycerides (Labrafil M-1944CS), PEG400, PEG 400 caprylic/capric glycerides (Labrasol), polyoxyl 40 stearate(PEG 1750 monosterate), polyoxyl 8 stearate (PEG 400 monosterate),polyvinyl pyrolidone, propylene carbonate, propylene glycol, solutolHS15, sorbitan monooleate (Span 20), sulfobutylether-beta-cyclodextrin,transcutol, triacetin, 1-dodecylazacyclo-heptan-2-one, caprolactam,castor oil, cottonseed oil, ethyl acetate, medium chain triglycerides,methyl acetate, oleic acid, safflower oil, sesame oil, soybean oil,tetrahydrofuran, glycerin, and PEG 4 kDa. The SBP may include silkfibroin, wherein the silk fibroin is present in the SBP at aconcentration of from about 0.1% (w/v) to about 30% (w/v). The SBP mayinclude from about 0.1% (w/v) to about 30% (w/v) of the oculartherapeutic agent. The SBP may include from about 0.1% (w/v) to about30% (w/v) of the excipient. The SBP may include an osmolarity of fromabout 275 mOsm to about 285 mOsm. The SBP may include a ratio of silkfibroin concentration (w/v) to excipient concentration (w/v) of fromabout 0.01 to about 0.5. The ratio of silk fibroin concentration (w/v)to excipient concentration (w/v) may be about 0.3. The SBP may include aratio of ocular therapeutic agent concentration (w/v) to silk fibroinconcentration (w/v) of from about 0.3 to about 4.2. The SBP may includea ratio of ocular therapeutic agent concentration (w/v) to excipientconcentration (w/v) of from about 0.1 to about 1. The ocular therapeuticagent may be a small molecule or a protein. The ocular therapeutic agentmay be a non-steroidal anti-inflammatory drug (NSAID). The NSAID mayinclude one or more of aspirin, carprofen, celecoxib, deracoxib,diclofenac, diflunisal, etodolac, fenoprofen, firocoxib, flurbirofen,ibuprofen, indomethacin, ketoprofen, ketorolac, mefenamic acid,meloxicam, nabumetone, naproxen, oxaprozin, piroxicam, robenacoxib,salsalate, sulindac, and tolmetin. The NSAID may be celecoxib. Theocular therapeutic agent may be a protein. The protein may be selectedfrom the group consisting of lysozyme, bovine serum albumin (BSA),bevacizumab, and VEGF-related agents. The SBP may be formulated forintraocular administration. The SBP may be formulated for one or more ofintravitreal administration, intraretinal administration, intracomealadministration, intrascleral administration, punctal administration,administration to the anterior sub-Tenon's, suprachoroidaladministration, administration to the posterior sub-Tenon's, subretinaladministration, administration to the fomix, administration to the lens,and intra-aqueous humor administration. The SBP may be biocompatible.The SBP may include a solution. The SBP may include a lyophilizedpowder. The SBP may stabilize the ocular therapeutic agent. The SBP maybe non-immunogenic. The SBP may include the composition of any of thesamples listed in any of Tables 1-41.

In some embodiments, the present disclosure provides a method oftreating a subject that includes contacting the subject with an SBPdescribed herein. The subject may have an ocular indication. The ocularindication may include inflammation. The ocular indication may includeone or more of an infection, refractive errors, age related maculardegeneration, cystoid macular edema, cataracts, diabetic retinopathy(proliferative and non-proliferative), glaucoma, amblyopia, strabismus,color blindness, cytomegalovirus retinitis, keratoconus, diabeticmacular edema (proliferative and non-proliferative), low vision, ocularhypertension, retinal detachment, eyelid twitching, inflammation,uveitis, bulging eyes, dry eye disease, floaters, xerophthalmia,diplopia, Graves' disease, night blindness, eye strain, red eyes,nystagmus, presbyopia, excess tearing, retinal disorder, conjunctivitis,cancer, comeal ulcer, corneal abrasion, snow blindness, scleritis,keratitis, Thygeson's superficial punctate keratopathy, comealneovascularization. Fuch's dystrophy, keratoconjunctivitis sicca,iritis, chorioretinal inflammation (e.g. chorioretinitis, choroiditis,retinitis, retinochoroiditis, pars planitis, Harada's disease, aniridia,macular scars, solar retinopathy, choroidal degeneration, choroidaldystrophy, choroideremia, gyrate atrophy, choroidal hemorrhage,choroidal detachment, retinoschisis, hypertensive retinopathy, Bull'seye maculopathy, epiretinal membrane, peripheral retinal degeneration,hereditary retinal dystrophy, retinitis pigmentosa, retinal hemorrhage,retinal vein occlusion, and separation of retinal layers. The SBP may beadministered via one or more of oral administration, intravenousadministration, topical administration, and ocular administration. TheSBP may be administered via one or more of intravitreal administration,intraretinal administration, intracomeal administration, intrascleraladministration, and intra-aqueous humor administration. The SBP may beadministered via intravitreal administration. The intravitrealadministration may include intravitreal injection. The intravitrealinjection may be performed by pushing a wire through a syringe andneedle or cannula loaded with the SBP. The wire may be pushed until thewire extends past the needle or cannula. The SBP may be used to deliverthe ocular therapeutic agent at a dose of from about 1 μg to about 5,000μg. The dose may be about 750 μg. Contacting the subject with the SBPmay result in a concentration of the ocular therapeutic agent in an eyeof the subject of from about 0.01 ng/mL to about 60,500 ng/mL. Theconcentration of ocular therapeutic agent in one or more components ofthe eye may be from about 0.01 ng/mL to about 60,500 ng/mL. The one ormore components of the eye may be selected from the group consisting ofaqueous humor, vitreous humor, retina, choroid, sclera, lens, fomix,conjunctiva, lacrimal punctum, capsule of Tenon, iris, pupal, cornea,ciliary muscle, fovea, optic nerve, macula, blood vessel, anteriorchamber, posterior chamber, and sub-tenon space. The concentration ofocular therapeutic agent in the aqueous humor may be from about 0.01ng/mL to about 2.0 ng/mL. The one or more components of the eye mayinclude the vitreous humor. The concentration of the ocular therapeuticagent in the vitreous humor may be from about 10 ng/mL to about 30,000ng/mL. The one or more components of the eye may include the retinaand/or choroid. The concentration of the ocular therapeutic agent in theretina and/or choroid may be from about 10 ng/mL to about 60,500 ng/mL.The ocular therapeutic agent may be detectable in one or more componentsof the eye for at least 3 months. The ocular therapeutic agent detectedmay remain at a steady level for at least 3 months. The oculartherapeutic agent may be detectable in one or more components of the eyefor at least 6 months. The ocular therapeutic agent detected may remainat a steady level for at least 6 months. Intraocular pressure may bereduced. The SBP may be well tolerated. The subject may be contactedwith a dose of the ocular therapeutic agent sufficient to achieve aconcentration of the ocular therapeutic agent in an eye of the subjector a component of an eye of the subject that is equal to or greater thanthe effective concentration for the ocular therapeutic agent. Theconcentration of the ocular therapeutic agent in the eye or eyecomponent may be greater than the effective concentration of the oculartherapeutic agent. The concentration of the ocular therapeutic agent inthe eye or eye component may be at least 1.5-fold greater than theeffective concentration of the ocular therapeutic agent. The eyecomponent may include the vitreous humor. The concentration of oculartherapeutic agent may be at least 1.5-fold greater than the effectiveconcentration of the ocular therapeutic agent. The eye component mayinclude the retina and/or choroid. The concentration of oculartherapeutic agent may be at least 4-fold greater than the effectiveconcentration of the ocular therapeutic agent. The SBP may be a rod. TheSBP may be a hydrogel.

In some embodiments, the present disclosure provides a method ofdelivering an ocular therapeutic agent to a subject by contacting an eyeof the subject with an SBP according to any of those described herein,wherein preparation of the SBP includes combining processed silk withthe ocular therapeutic agent. The SBP may be prepared as a rod. Thedensity of the SBP may be modulated by the concentration of processedsilk. The SBP may be prepared by extrusion through a tube. The tube maybe a needle. Extrusion may be carried out using a syringe. The SBP maybe incubated at approximately 37° C. The SBP may be incubated for up toapproximately 24 hours. The SBP may form a gel in the tube. The oculartherapeutic agent may be delivered to the subject's eye by release fromthe SBP while in contact with the subject's eye. Release of the oculartherapeutic agent from the SBP may be modulated by one or more of silkfibroin concentration, silk fibroin molecular weight. SBP volume, methodused to dry the SBP, ocular therapeutic agent molecular weight, andinclusion of at least one excipient. The technique used to dry the SBPmay include one or more of oven drying, lyophilization, and air drying.The SBP may include ocular therapeutic agent and silk fibroin at a w/wratio of from about 1 to about 4.2. Release of the ocular therapeuticagent from the SBP may occur at a rate that includes an initial burst.From about 0.1% to about 100% of the ocular therapeutic agent may bereleased from the SBP during an initial release period associated withthe initial burst. The rate of release of the therapeutic agent may beinversely related to the concentration of processed silk. The SBP may bea rod, and the amount of therapeutic agent released during the initialperiod associated with the initial burst may be inversely related to thedensity of the rod. Release of the ocular therapeutic agent from the SBPmay include a daily release percentage of from about 0.1% (w/w) to about5% (w/v). The SBP may be a rod, and the daily release percentage may beinversely related to the density of the rod. From about 1% to about 100%of the ocular therapeutic agent may be released from the SBP during arelease period of from about 1 day to about 10 months. The releaseperiod may begin upon contacting an eye of the subject with the SBP. Therelease period may be from about 1 day to about 5 months. The SBP may bea rod, and the release period may be proportional to the density of therod. From about 3% to about 100% of the ocular therapeutic agent may bereleased from the SBP over the release period.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages will beapparent from the following description of particular embodiments of theinvention, as illustrated in the accompanying drawings. The drawings arenot necessarily to scale; emphasis instead being placed uponillustrating the principles of various embodiments of the invention.

FIG. 1A is a scanning electron microscope (SEM) image showing a silkfibroin rod formulated with celecoxib.

FIG. 1B is a scanning electron microscope (SEM) image showing a silkfibroin rod formulated with celecoxib.

FIG. 1C is a scanning electron microscope (SEM) image showing a silkfibroin rod formulated with celecoxib.

FIG. 1D is a scanning electron microscope (SEM) image showing a silkfibroin rod formulated with celecoxib.

FIG. 2A is an image showing a silk fibroin rod formulated withcelecoxib, with a diameter of 430 μm.

FIG. 2B is a SEM image showing a silk fibroin rod formulated withcelecoxib, with a diameter of 430 μm.

FIG. 2C is a SEM image showing a silk fibroin rod formulated withcelecoxib, with a diameter of 430 μm.

FIG. 2D is a SEM image showing a silk fibroin rod formulated withcelecoxib, with a diameter of 430 μm.

FIG. 3 is a graph showing TNF-α concentration in human whole blood afteradministration of various concentrations of lipopolysaccharide (LPS) orsilk fibroin.

FIG. 4 is a plot of the cumulative release percentage of an API,celecoxib, over time for a hydrogel and a suspension of celecoxib.

DETAILED DESCRIPTION

Embodiments of the present disclosure relate to silk-based products(SBPs) and their methods of use. The term “silk” generally refers to afibrous material formed by insects and some other species that includestightly bonded protein filaments. Herein, the term “silk” is used in thebroadest sense and may embrace any forms, variants, or derivatives ofsilk discussed.

Silk fibers from silkworm moth (Bombyx mori) cocoons include two maincomponents, sericin (usually present in a range of 20-30%) and silkfibroin (usually present in a range of 70-80%). While not wishing to bebound by theory, structurally silk fibroin forms the center of the silkfibers and sericin acts as the gum coating the fibers. Sericin is agelatinous protein that holds silk fibers together with many of thecharacteristic properties of silk (see Qi et al. (2017) Int J Mol Sci18:237 and Deptuch et al. (2017) Materials 10:1417, the contents of eachof which are herein incorporated by reference in their entireties). Silkfibroin is an insoluble fibrous protein consisting of layers ofantiparallel beta sheets. Its primary structure mainly consists ofrecurrent serine, alanine, and glycine repeating units and theisoelectric point of silk fibroin has been determined to be around 4.2.Silk fibroin monomers include a complex of heavy chain (around 350 kDa)and light chain (around 25 kDa) protein components. Typically, thechains are joined by a disulfide bond. With some forms, heavy chain andlight chain segments are non-covalently bound to a glycoprotein, p25.Polymers of silk fibroin monomers may form through hydrogen bondingbetween monomers, typically increasing mechanical strength (see Qi etal. (2017) Int J Mol Sci 18:237). During silk processing, fragments ofsilk fibroin monomers may be produced, including, but not limited to,fragments of heavy and/or light chains. These fragments may retain theability to form hydrogen bonds with silk fibroin monomers and fragmentsthereof. Herein, the term “silk fibroin” is used in its broadest senseand embraces silk fibroin polymers, silk fibroin monomers, silk fibroinheavy and light chains, silk fibroin fragments, and variants,derivatives, or mixtures thereof from any of the wild type, geneticallymodified, or synthetic sources of silk described herein.

The present disclosure includes methods of preparing processed silk andSBPs, different forms of SBPs, and a variety of applications forutilizing processed silk and SBPs alone or in combination with variouscompounds, compositions, and devices.

1. Silk-Based Products

As used herein, the term “silk-based product” or “SBP” refers to anycompound, mixture, or other entity that is made up of or that iscombined with processed silk. “Processed silk,” as used herein, refersto any forms of silk harvested, obtained, synthesized, formatted,manipulated, or altered through at least one human intervention. SBPsmay include a variety of different formats suited for a variety ofdifferent applications. Examples of SBP formats include, but are notlimited to, fibers, nanofibers, implants, rods, gels, hydrogels, andsolutions. Additional formats are described herein. SBPs may findutility in variety of fields and for a variety of applications. Suchutility may be due to the unique physical and chemical properties ofsilk. These physical and chemical properties include, but are notlimited to, biocompatibility, biodegradability, bioresorbability,solubility, crystallinity, porosity, mechanical strength, thermalstability, and transparency. In some embodiments, SBPs may be used forone or more therapeutic applications. Such SBPs may include processedsilk, wherein the processed silk is or is derived from one or more ofraw silk, silk fibers, silk fibroin, and silk fibroin fragments.Processed silk present is some SBPs may include one or more silk fibroinpolymers, silk fibroin monomers, and/or silk fibroin fragments. In someembodiments, silk fibroin fragments include silk fibroin heavy chainfragments and/or silk fibroin light chain fragments. Some silk fibroinpresent in SBPs include a plurality of silk fibroin fragments. Each ofthe plurality of silk fibroin fragments may have a molecular weight offrom about 1 kDa to about 350 kDa. As a non-limiting example, the silkfibroin fragment may have a molecular weight of 1 kDa, 2 kDa, 3 kDa, 4kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa,30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa,75 kDa, 80 kDa, 85 kDa, 90 kDa, 95 kDa, 100 kDa, 105 kDa, 110 kDa, 115kDa, 120 kDa, 125 kDa, 130 kDa, 135 kDa, 140 kDa, 145 kDa, 150 kDa, 155kDa, 160 kDa, 165 kDa, 170 kDa, 175 kDa, 180 kDa, 185 kDa, 190 kDa, 195kDa, 200 kDa, 205 kDa, 210 kDa, 215 kDa, 220 kDa, 225 kDa, 230 kDa, 235kDa, 240 kDa, 245 kDa, 250 kDa, 255 kDa, 260 kDa, 265 kDa, 270 kDa, 275kDa, 280 kDa, 285 kDa, 290 kDa, 295 kDa, 300 kDa, 305 kDa, 310 kDa, 315kDa, 320 kDa, 325 kDa, 330 kDa, 335 kDa, 340 kDa, 345 kDa, or 350 kDa.As a non-limiting example, the silk fibroin fragment may have amolecular weight of 1-5 kDa, 1-10 kDa, 1-15 kDa, 1-25 kDa, 1-50 kDa,1-75 kDa, 1-100 kDa, 1-150 kDa, 1-200 kDa, 1-250 kDa, 1-300 kDa, 1-350kDa, 5-10 kDa, 5-15 kDa, 5-25 kDa, 5-50 kDa, 5-75 kDa, 5-100 kDa, 5-150kDa, 5-200 kDa, 5-250 kDa, 5-300 kDa, 5-350 kDa, 10-15 kDa, 10-25 kDa,10-50 kDa, 10-75 kDa, 10-100 kDa, 10-150 kDa, 10-200 kDa, 10-250 kDa,10-300 kDa, 10-350 kDa, 15-25 kDa, 15-50 kDa, 15-75 kDa, 15-100 kDa,15-150 kDa, 15-200 kDa, 15-250 kDa, 15-300 kDa, 15-350 kDa, 25-50 kDa,25-75 kDa, 25-100 kDa, 25-150 kDa, 25-200 kDa, 25-250 kDa, 25-300 kDa,25-350 kDa, 50-75 kDa, 50-100 kDa, 50-150 kDa, 50-200 kDa, 50-250 kDa,50-300 kDa, 50-350 kDa, 75-100 kDa, 75-150 kDa, 75-200 kDa, 75-250 kDa,75-300 kDa, 75-350 kDa, 100-150 kDa, 100-200 kDa, 100-250 kDa, 100-300kDa, 100-350 kDa, 150-200 kDa, 150-250 kDa, 150-300 kDa, 150-350 kDa,200-250 kDa, 200-300 kDa, 200-350 kDa, 250-300 kDa, 250-350 kDa, and300-350 kDa.

Sources of Silk

SBPs may include processed silk obtained from any one of a variety ofsources. Processed silk may include raw silk. “Raw silk,” as usedherein, refers to silk that has been harvested, purified, isolated, orotherwise collected from silk producers. The term “silk producer,” asused herein, refers to any organism capable of producing silk. Raw silkhas been processed in large quantities for thousands of years, primarilyfrom silkworms (Bombyx mori), which use silk to form their cocoon. Rawsilk from silkworm cocoons includes silk fibroin and sericin that issecreted onto silk fibroin during cocoon formation. Raw silk may beharvested as a silk fiber. As used herein, the term “silk fiber” refersto any silk that is in the form of a filament or thread. Silk fibers mayvary in length and width and may include, but are not limited to, yarns,strings, threads, and nanofibers. In some embodiments, raw silk may beobtained in the form of a yarn.

Silk Producers

In some embodiments, processed silk includes silk obtained from a silkproducer. There are many species of silk producers in nature capable ofproducing silk. Silk producers may be insect species, such as silkworms.Some silk producers include arachnid species. In some embodiments, silkproducers include species of mollusk. Silk produced by different silkproducing species may vary in physical and/or chemical properties. Suchproperties may include amino acid content, secondary structure (e.g.β-sheet content), mechanical properties (e.g. elasticity), and others.

In some embodiments, processed silk may be obtained from the silkwormspecies Bombyx mori. Other examples of silk producer species include,but are not limited to, Bombyx mandarina, Bombyx sinesis, Anaphemoloneyi, Anaphe panda, Anaphe reticulate, Anaphe ambrizia, Anaphecarteri, Anaphe venata, Anapha infracta, Antheraea assamensis, Antheraeaassama, Antheraea mylitta, Antheraea pernyi, Antheraeayamamai, Antheraeapolyphemus, Antheraea oculea, Anisota senatoria, Apis mellifera, Araneusdiadematus, Araneus cavaticus, Automeris io, Atticus atlas, Copaxamultifenestrata, Coscinocera hercules, Callosamia promethea, Eupackardiacalleta, Eurprosthenops australis, Gonometa postica, Gonometarufobrunnea, Hyalophora cecropia, Hvalophora euryalus, Halophoragloveri, Airanda auretia, Nephila madagascarensis, Nephila clavipes,Pachypasa olus, Pachypasa atus, Philosamia ricini. Pinna squamosa,Rothschildia hesperis, Rothschildia lebeau, Samia Cynthia, and Samiaricini.

Silk Properties

In some embodiments, processed silk may be selected based on or preparedto include features affecting one or more properties of the processedsilk. Such properties may include, but are not limited to, stability,complex stability, composition stability, payload retention or release,payload release rate, wettability, mechanical strength, tensilestrength, elongation capabilities, elasticity, compressive strength,stiffness, shear strength, toughness, torsional stability, temperaturestability, moisture stability, strength, flexibility, solubility,crystallinity, viscosity, density, thickness, and porosity. Featuresaffecting one or more processed silk properties may include silksecondary structure. Secondary structure refers to three-dimensionalarrangements of polypeptide chains based on local interactions betweenneighboring residues. Common secondary structures include β-pleatedsheets and α-helices. Silk secondary structure may enhance or attenuatesolubility. In some embodiments, β-sheet secondary structure content mayenhance processed silk crystallinity. “Crystallinity” refers to thedegree of structure and arrangement between atoms or molecules in acompound, with increased structure yielding greater crystallinity.β-sheet structures may be antiparallel β-sheets. In some embodiments,processed silk includes polypeptides with random coil secondarystructure. Some processed silk includes polypeptides with coiled coilsecondary structure. In some embodiments, processed silk includes acombination of two or more forms of secondary structure. In someembodiments, processed silk may include polypeptides with multiplerepeats. As used herein when referring to polypeptides, the term“multiple repeat” refers to an amino acid sequence that is duplicatedtwo or more times in succession within a polypeptide. Silk fibroin heavychains include multiple repeats that enable static interactions betweenparallel silk fibroin heavy chains. Multiple repeats may include repeatsof the sequences GAGAGS (SEQ ID NO: 1) and/or GA. In some embodiments,the A of GA dipeptides may be replaced with S or Y. In some embodiments,multiple repeats may include any of those presented in Qi et al. (2017)Int J Mol Sci 18:237, the contents of which are herein incorporated byreference in their entirety. Multiple repeats may enable formation ofstable, crystalline regions of antiparallel β-sheets.

Processed silk may include silk fibroin forms described by Qi et al.(2017) Int J Mol Sci 18:237 and Cao et al. (2009) Int J Mol Sci10:1514-1524, the contents of each of which are herein incorporated byreference in their entirety. These silk fibroin forms are referred to assilk I, silk II, and silk III. Silk I and silk II forms are commonlyfound in nature. Silk I predominantly includes random coil secondarystructures. Silk II predominantly includes p-sheet secondary structure.Silk III predominantly includes an unstable structure.

Processed silk may be treated to modulate β-sheet content and/orcrystallinity. In some embodiments these treatments are used to reducethe solubility of the silk fibroin or silk fibroin composition.Treatments may include, but are not limited to, alteration of the pH,sonication of the silk fibroin, incorporation of an excipient,increasing or decreasing the temperature, treatment with acid, treatmentwith formic acid, treatment with glycerol, treatment with an alcohol,treatment with methanol, treatment with ethanol, treatment withisopropanol, and/or treatment with a mixture of alcohol and water. Insome embodiments, treatments result in transition between forms of silkI, II, or III. Such methods may include any of those described in Cao etal. (2009) Int J Mol Sci 10:1514-1524).

Porosity

In some embodiments, processed silk may include variations in porosity.As used herein, the term “porosity” refers to the frequency with whichholes, pockets, channels, or other spaces occur in a material, in somecases influencing the movement of elements to and/or from the material.Processed silk porosity may influence one or more other silk propertiesor properties of an SBP that includes the processed silk. Theseproperties may include, but are not limited to, stability, payloadretention or release, payload release rate, wettability, mechanicalstrength, tensile strength, elongation capabilities, density, thickness,elasticity, compressive strength, stiffness, shear strength, toughness,torsional stability, temperature stability, and moisture stability. Insome embodiments, processed silk porosity may control the diffusion ortransport of agents from, within, or into the processed silk or SBP.Such agents may include, but are not limited to, therapeutics,biologics, chemicals, small molecules, oxidants, antioxidants,macromolecules, microspheres, nanospheres, cells, or any payloadsdescribed herein.

Processed silk porosity may be modulated during one or more processingsteps or during fabrication of an SBP (e.g., see InternationalPublication No. WO2014125505 and U.S. Pat. No. 8,361,617, the contentsof each of which are herein incorporated by reference in theirentirety). In some embodiments, processed silk porosity may be modulatedby one or more of sonication, centrifugation, modulating silk fibroinconcentration, modulating salt concentration, modulating pH, modulatingsecondary structural formats, applying shear stress, modulatingexcipient concentration, chemical modification, crosslinking, orcombining with cells, bacteria, and/or viral particles.

Strength and Stability

Processed silk strength and stability are important factors for manyapplications. In some embodiments, processed silk may be selected basedon or prepared to maximize mechanical strength, tensile strength,elongation capabilities, elasticity, flexibility, compressive strength,stiffness, shear strength, toughness, torsional stability, biologicalstability, resistance to degradation, and/or moisture stability. In someembodiments, processed silk had a non-acidic microenvironment. In someembodiments, the non-acidic microenvironment enhances the stability ofprocessed silk and or SBPs. In some embodiments, the non-acidicmicroenvironment enhances the stability of therapeutic agents formulatedwith the processed silk and/or SBP. In some embodiments, the tensilestrength of processed silk is stronger than steel. In some embodiments,the tensile strength of an SBP is stronger than steel.

Biocompatibility

In some embodiments, processed silk may be selected based on or preparedto maximize biocompatibility. As used herein, the term“biocompatibility” refers to the degree with which a substance avoidsprovoking a negative biological response in an organism exposed to thesubstance. The negative biological response may include an inflammatoryresponse, local sensitization, hemorrhage, and/or other complicationsknown to those skilled in the art. In some embodiments, administrationof processed silk or an SBP does not induce an inflammatory response,local sensitization, hemorrhage, and/or other complications known tothose skilled in the art. In some embodiments, contact with processedsilk or an SBP does not induce an inflammatory response, localsensitization, hemorrhage, and/or other complications known to thoseskilled in the art. In some embodiments, processed silk biocompatibilityis enhanced through preparations that produce only non-toxic byproductsduring degradation. In some embodiments, exposure to an SBP generates atolerable biological response, within an acceptable threshold known tothose skilled in the art. In some embodiments, processed silk isbiocompatible in humans and human whole blood. In some embodiments,processed silk is biocompatible in animals. In some embodiments,processed silk produces no adverse reactions, no acute inflammation, andno immunogenicity in vivo. In some embodiments, the processed silk orSBP is safe to use in vivo. In some embodiments, processed silk or SBPsare biocompatible and/or tolerable in vitro. In some embodiments,processed silk or SBPs are biocompatible and/or tolerable in vivo. Insome embodiments, no inflammatory response, local sensitization,hemorrhage, and/or other complications occur after up 1 day, up to 3days, up to 1 week, up to I month, up to 3 months, up to 4 months, up to6 months, up to 7 months, or up to 1 year of contact with processed silkor an SBP.

Biodegradability

In some embodiments, processed silk may be selected based on or preparedto maximize biodegradability. As used herein, the term“biodegradability” refers to the degree with which a substance avoidsprovoking a negative response to an environment exposed to the substanceas it deteriorates. The negative environmental response may include aresponse to toxic byproducts generated as a substance deteriorates. Insome embodiments, processed silk biodegradability is enhanced throughpreparations that produce only non-toxic byproducts during degradation.In some embodiments, processed silk biodegradability is enhanced throughpreparations that produce only inert amino acid byproducts. In someembodiments, the SBP and/or SBP by products are considered naturallyderived and environmentally and/or eco-friendly.

Anti-Evaporative Properties

In some embodiments, processed silk may be selected based on or preparedto reduce the evaporation of a solution. In some embodiments, processedsilk may reduce the evaporation of a solution. In some embodiments, anSBP may demonstrate anti-evaporative properties by creating a waterbarrier. In some embodiments, processed silk may extend the lifetime orresidence time of an SBP product due to its ability to preventevaporation. In some embodiments, processed silk may increase the amountof time required for a solution to evaporate. In some embodiments,processed silk may be selected based on or prepared to reduce theevaporation of a solution. In some embodiments, processed silk mayreduce the evaporation of a solution. In some embodiments, processedsilk may extend the lifetime or residence time of an SBP product due toits ability to prevent evaporation. In some embodiments, processed silkmay increase the amount of time required for a solution to evaporate.

Anti-Inflammatory Properties

In some embodiments, processed silk or SBPs may have or be prepared tomaximize anti-inflammatory properties. It has been reported that silkfibroin peptide derived from silkworm Bombyx mori exhibitedanti-inflammatory activity in a mice model of inflammation (Kim et al.,(2011) BMB Rep 44(12):787-92; the contents of which are incorporated byreference in their entirety). In some embodiments, processed silk orSBPs may be administered to a subject alone or in combination with othertherapeutic agents to elicit anti-inflammatory effects. It iscontemplated that processed silk or SBPs alone or combination with othertherapeutic agents may be used to treat various inflammatory diseases.For example, processed silk or SBPs may reduce signs and symptoms ofinflammation, such as but not limited to, swelling, redness, tenderness,rashes, fever, and pain.

Processed Silk and Related Methods

Various processing methods may be used to obtain specific forms orformats of processed silk. Such processing methods may include, but arenot limited to, acidifying, air drying, alkalinizing, annealing,autoclaving, chemical crosslinking, chemical modification,concentration, cross-linking, degumming, dissolving, dry spinning,drying, electrifying, electrospinning, electrospraying, emulsifying,encapsulating, extraction, extrusion, gelation, harvesting, heating,lyophilization, molding, oven drying, pH alteration, precipitation,purification, shearing, sonication, spinning, spray drying, sprayfreezing, spraying, vapor annealing, vortexing, and water annealing. Theprocessing steps may be used to prepare final SBPs or they may be usedto generate processed silk preparations. As used herein, the term“processed silk preparation” is generally used to refer to processedsilk or compositions that include processed silk that are prepared foror obtained during or after one or more processing steps. Processed silkpreparations may be SBPs, may be components of SBPs, or may be used as astarting or intermediate composition in the preparation of SBPs.Processed silk preparations may include other components related toprocessing (e.g., solvents, solutes, impurities, catalysts, enzymes,intermediates, etc.). Processed silk preparations that include silkfibroin may be referred to as silk fibroin preparations. In someembodiments, processed silk manufacturing is simple, scalable, and/orcost effective.

In some embodiments, processed silk may be prepared as, provided as, orincluded in a yarn, thread, string, a nanofiber, a particle, ananoparticle, a microsphere, a nanosphere, a powder, a solution, a gel,a hydrogel, an organogel, a mat, a film, a foam, a membrane, a rod, atube, a patch, a sponge, a scaffold, a capsule, an excipient, animplant, a solid, a coating, and/or a graft.

In some embodiments, the formulations are prepared to be sterile. Asused herein, the term “sterile” refers to something that is aseptic. Insome embodiments, SBPs are prepared from sterile materials. In someembodiments, SBPs are prepared and then sterilized. In some embodiments,processed silk is degummed and then sterilized. In some embodiments,processed silk is sterilized and then degummed. Processed silk and/orSBPs may be sterilized via gamma radiation, autoclave (e.g., autoclavesterilization), filtration, electron beam, and any other method known tothose skilled in the art.

In some embodiments, processed silk may be stored frozen or dried to astable soluble form. Processed silk may be frozen with cryoprotectants.Cryoprotectants may include, but are not limited to, phosphate buffer,sucrose, trehalose, histidine, and any other cryoprotectant known to oneof skill in the art. In some embodiments, SBPs may be stored frozen ordried to a stable soluble form. In some embodiments, the SBPs may besolutions.

Harvesting Silk

In some embodiments, processed silk is harvested from silk producercocoons. Cocoons may be prepared by cultivating silkworm moths andallowing them to pupate. Once fully formed, cocoons may be treated tosoften sericin and allow for unwinding of the cocoon to form raw silkfiber. The treatment may include treatment with hot air, steam, and/orboiling water. Raw silk fibers may be produced by unwinding multiplecocoons simultaneously. The resulting raw silk fibers include both silkfibroin and sericin. Subsequent processing may be carried out to removesericin from the raw silk fibers or from later forms of processed silkor SBPs. In some embodiments, raw silk may be harvested directly fromthe silk glands of silk producers. Raw silk may be harvested from wildtype or GMO silk producers.

Extraction of Sericin/Degumming

In some embodiments, sericin may be removed from processed silk, aprocess referred to herein as “degumming.” The processed silk mayinclude raw silk, which includes sericin secreted during cocoonformation. Methods of degumming may include heating (e.g., boiling) in adegumming solution. As used herein, the term “degumming solution” refersto a composition used for sericin removal that includes at least onedegumming agent. As used herein, a “degumming agent” refers to anysubstance that may be used for sericin removal. Heating in degummingsolution may reduce or eliminate sericin from processed silk. In someembodiments, heating in degumming solution includes boiling. Heating indegumming solution may be followed by rinsing to enhance removal ofsericin that remains after heating. In some embodiments, raw silk isdegummed before further processing or utilization in SBPs. In otherembodiments, raw silk is further processed or otherwise incorporatedinto an SBP prior to degumming. Such methods may include any of thosepresented in European Patent No. EP2904134 or United States PublicationNo. US2017031287, the contents of each of which are herein incorporatedby reference in their entirety.

Degumming agents and/or degumming solution may include, but are notlimited to water, alcohols, soaps, acids, alkaline solutions, and enzymesolutions. In some embodiments, degumming solutions may includesalt-containing alkaline solutions. Such solutions may include sodiumcarbonate. Sodium carbonate concentration may be from about 0.01 M toabout 0.3 M. In some embodiments, sodium carbonate concentration may befrom about 0.01 M to about 0.05 M, about 0.05 M to about 0.1 M, fromabout 0.1 M to about 0.2 M. or from about 0.2 M to about 0.3 M. In someembodiments, sodium carbonate concentration may be 0.02 M. In someembodiments, degumming solutions may include from about 0.01% to about1% (w/v) sodium carbonate. In some embodiments, degumming solutions mayinclude from about 0.01% to about 10% (w/v) sodium carbonate. In someembodiments, degumming solutions may include from about 0.01% (w/v) toabout 1% (w/v), from about 1% (w/v) to about 2% (w/v), from about2^(%)(w/v) to about 3% (w/v), from about 3% (w/v) to about 4% (w/v),from about 4% (w/v) to about 5% (w/v), or from about 5% (w/v) to about10% (w/v) sodium carbonate. In some embodiments, degumming solutions mayinclude sodium dodecyl sulfate (SDS). Such degumming solutions mayinclude any those described in Zhang et al. (2012) J Translational Med10:117, the contents of which are herein incorporated by reference intheir entirety. In some embodiments, degumming solutions include boricacid. Such solutions may include any of those taught in European PatentNo. EP2904134, the contents of which are herein incorporated byreference in their entirety. In some embodiments, the degumming solutionmay have a pH of from about 0 to about 5, from about 2 to about 7, fromabout 4 to about 9, from about 5 to about 11, from about 6 to about 12,from about 6.5 to about 8.5, from about 7 to about 10, from about 8 toabout 12, and from about 10 to about 14. In some embodiments, processedsilk may be present in degumming solutions at concentrations of fromabout 0.1% to about 2%, from about 0.5% to about 3%, from about 1% toabout 4%, or from about 2% to about 5% (w/v). In some embodiments,processed silk is present in degumming solutions at concentrations ofgreater than 5% (w/v).

Degumming may be carried out by boiling in degumming solutions at ornear (e.g., within about 5% of) atmospheric boiling temperatures. Someboiling temperatures may be from about 60° C. to about 115° C. In someembodiments, boiling is carried out at 100° C. In some embodiments,boiling is carried out at about 90° C., about 91° C., about 92° C. about93° C., about 94° C., about 95° C., about 96° C., about 97° C., about98° C., about 99° C., about 100° C., about 101° C., about 102° C., about103° C., about 104° C., about 105° C., about 106° C., about 107° C.,about 108° C., about 109° C., or about 110° C.

In some embodiments, degumming includes heating in degumming solutionfor a period of from about 10 seconds to about 45 seconds, from about 30seconds to about 90 seconds, from about 1 min to about 5 min, from about2 min to about 10 min, from about 5 min to about 15 min, from about 10min to about 25 min, from about 20 min to about 35 min, from about 30min to about 50 min, from about 45 min to about 75 min, from about 60min to about 95 min, from about 90 min to about 125 min, from about 120min to about 175 min, from about 150 min to about 200 min, from about180 min to about 250 min, from about 210 min to about 350 min, fromabout 240 min to about 400 min, from about 270 min to about 450 min,from about 300 min to about 500 min, from about 330 min to about 550min, from about 360 min to about 600 min, from about 390 min to about700 min, from about 420 min to about 800 min, from about 450 min toabout 900 min, from about 480 min to about 1000 min, from about 510 minto about 1100 min, from about 540 min to about 1200 min, from about 570min to about 1300 min, from about 600 min to about 1400 min, from about630 min to about 1500 min, from about 660 min to about 1600 min, fromabout 690 min to about 1700 min, from about 720 min to about 1800 min,from about 1440 min to about 1900 min, from about 1480 min to about 2000min, or longer than 2000 min.

In some embodiments, processed silk preparations may be characterized bythe number of minutes boiling was carried out for preparation, a valuereferred to herein as “minute boil” or “mb.” The minute boil value of apreparation may be associated with known or presumed characteristics ofsimilar preparations with the same minute boil value. Suchcharacteristics may include concentration and/or molecular weight ofpreparation compounds, proteins, or protein fragments altered duringboiling. In some embodiments, processed silk preparations (e.g., silkfibroin preparations) have an mb value of from about 1 mb to about 5 mb,from about 2 mb to about 10 mb, from about 5 mb to about 15 mb, fromabout 10 mb to about 25 mb, from about 20 mb to about 35 mb, from about30 mb to about 50 mb, from about 45 mb to about 75 mb, from about 60 mbto about 95 mb, from about 90 mb to about 125 mb, from about 120 mb toabout 175 mb, from about 150 mb to about 200 mb, from about 180 mb toabout 250 mb, from about 210 mb to about 350 mb, from about 240 mb toabout 400 mb, from about 270 mb to about 450 mb, from about 300 mb toabout 480 mb, or greater than 480 mb.

In some embodiments, degumming is carried out by treatment with hightemperatures and/or pressures. Such methods may include any of thosepresented in International Publication No. WO2017200659, the contents ofwhich are herein incorporated by reference in their entirety.

Processed Silk Preparation Characterization

Preparations of processed silk may include mixtures of silk fibroinpolymers, silk fibroin monomers, silk fibroin heavy chains, silk fibroinlight chains, sericin, and/or fragments of any of the foregoing. Wherethe exact contents and ratios of components in such processed silkpreparations are unknown, the preparations may be characterized by oneor more properties of the preparation or by conditions or methods usedto obtain the preparations.

Solubility and Concentration

Processed silk preparations may include solutions that include processedsilk (also referred to herein as “processed silk solutions”). Processedsilk solutions may be characterized by processed silk concentration. Forexample, processed silk may be dissolved in a solvent after degumming togenerate a processed silk solution of silk fibroin for subsequent use.Solvent used to dissolve processed silk may be a buffer. In someembodiments, solvent used is an organic solvent. Organic solvents mayinclude, but are not limited to hexafluoroisopropanol (HFIP), methanol,isopropanol, ethanol, or combinations thereof. In some embodiments,solvents include a mixture of an organic solvent and water or an aqueoussolution. Solvents may include water or aqueous solutions. Aqueoussolutions may include aqueous salt solutions that include one or moresalts. Such salts may include but are not limited to lithium bromide(LiBr), lithium thiocyanate, Ajisawa's reagent, a chaotropic agent,calcium nitrate, or other salts capable of solubilizing silk, includingany of those disclosed in U.S. Pat. No. 9,623,147 (the content of whichis herein incorporated by reference in its entirety). In someembodiments, solvents used in processed silk solutions may includeAjisawa's reagent, as described in Zheng et al. (2016) Journal ofBiomaterials Applications 31:450-463, the content of which is hereinincorporated by reference in its entirety. Ajisawa's reagent comprises amixture of calcium chloride, ethanol, and water in a molar ratio of1:2:8 respectively. In some embodiments, solvents used in processed silksolutions include high salt solutions. In some embodiments, the solutioncomprises 5 to 13 M LiBr. The concentration of LiBr may be 9.3 M.

In some embodiments, processed silk is present in processed silksolutions at a concentration of from about 0.01% (w/v) to about 1%(w/v), from about 0.05% (w/v) to about 2^(%) (w/v), from about 1% (w/v)to about 5% (w/v), from about 2% (w/v) to about 10% (w/v), from about 4%(w/v) to about 16% (w/v), from about 5% (w/v) to about 20% (w/v), fromabout 8% (w/v) to about 24% (w/v), from about 10% (w/v) to about 30%(w/v), from about 12% (w/v) to about 32% (w/v), from about 14% (w/v) toabout 34% (w/v), from about 16% (w/v) to about 36% (w/v), from about 18%(w/v) to about 38% (w/v), from about 20% (w/v) to about 40% (w/v), fromabout 22% (w/v) to about 42% (w/v), from about 24% (w/v) to about 44%(w/v), from about 26% (w/v) to about 46% (w/v), from about 28% (w/v) toabout 48% (w/v), from about 30% (w/v) to about 50% (w/v), from about 35%(w/v) to about 55% (w/v), from about 40% (w/v) to about 60% (w/v), fromabout 45% (w/v) to about 65% (w/v), from about 50% (w/v) to about 70%(w/v), from about 55% (w/v) to about 75% (w/v), from about 60% (w/v) toabout 80% (w/v), from about 65% (w/v) to about 85% (w/v), from about 70%(w/v) to about 90% (w/v), from about 75% (w/v) to about 95% (w/v), fromabout 80% (w/v) to about 96% (w/v), from about 85% (w/v) to about 97%(w/v), from about 90% (w/v) to about 98% (w/v), from about 95% (w/v) toabout 99% (w/v), from about 96% (w/v) to about 99.2% (w/v), from about97% (w/v) to about 99.5% (w/v), from about 98% (w/v) to about 99.8%(w/v), from about 99% (w/v) to about 99.9% (w/v), or greater than 99.9%(w/v). In some embodiments, the processed silk is silk fibroin.

Processed silk solutions may be characterized by the length of timeand/or temperature needed for processed silk to dissolve. The length oftime used to dissolve processed silk in solvent is referred to herein as“dissolution time.” Dissolution times for dissolution of processed silkin various solvents may be from about 1 min to about 5 min, from about 2min to about 10 min, from about 5 min to about 15 min, from about 10 minto about 25 min, from about 20 min to about 35 min, from about 30 min toabout 50 min, from about 45 min to about 75 min, from about 60 min toabout 95 min, from about 90 min to about 125 min, from about 120 min toabout 175 min, from about 150 min to about 200 min, from about 180 minto about 250 min, from about 210 min to about 350 min, from about 240min to about 360 min, from about 270 min to about 420 min, from about300 min to about 480 min, or longer than 480 minutes.

The temperature used to dissolve processed silk in solvent is referredto herein as “dissolution temperature.” Dissolution temperatures usedfor dissolution of processed silk in solvent may include roomtemperature. In some embodiments, dissolution temperature may be fromabout 0° C. to about 10° C., from about 4° C. to about 25° C., fromabout 20° C. to about 35° C., from about 30° C. to about 45° C., fromabout 40° C. to about 55° C., from about 50° C. to about 65° C., fromabout 60° C. to about 75° C., from about 70° C. to about 85° C., fromabout 80° C. to about 95° C., from about 90° C. to about 105° C., fromabout 100° C. to about 115° C., from about 110° C. to about 125° C.,from about 120° C. to about 135° C., from about 130° C. to about 145°C., from about 140° C. to about 155° C., from about 150° C. to about165° C., from about 160° C. to about 175° C., from about 170° C. toabout 185° C., from about 180° C. to about 200° C., or greater than 200°C. In some embodiments, the processed silk is silk fibroin. Dissolutionof some processed silk solutions may use a dissolution temperature of60° C. Dissolution of some processed silk solutions may use adissolution temperature of 80° C., as described in Zheng et al. (2016)Journal of Biomaterials Applications 31:450-463. In some embodiments,dissolution includes boiling. In some embodiments, dissolution may becarried out by autoclaving. In some embodiments, silk fibroin solutionsmay be prepared according to any of the methods described inInternational Publication No. WO2017200659 or Abdel-Naby (2017) PLoS One12(11):e0188154), the contents of each of which are herein incorporatedby reference in their entirety.

In some embodiments, one or more of sucrose, phosphate buffer, trisbuffer, trehalose, mannitol, citrate buffer, ascorbate, histidine,and/or a cryoprotective agent is added to processed silk solutions.

Chaotropic Agents

In some embodiments, processed silk may be dissolved with the aid of achaotropic agent. As used herein, a “chaotropic agent” refers to asubstance that disrupts hydrogen bonding networks in aqueous solutionsto facilitate dissolution of a solute. Chaotropic agents typicallymodify the impact of hydrophobicity on dissolution. Chaotropic agentsmay be organic compounds. Such compounds may include, but are notlimited to, sodium dodecyl sulfate, ethanol, methanol, phenol,2-propanol, thiourea, urea, n-butanol, and any other chemicals capableof solubilizing silk. In some embodiments, the chaotropic agent is asalt, including, but not limited to, zinc chloride, calcium nitrate,lithium perchlorate, lithium acetate, sodium thiocyanate, calciumthiocyanate, magnesium thiocyanate, calcium chloride, magnesiumchloride, guanidinium chloride, lithium bromide, lithium thiocyanate,copper salts, and other salts capable of solubilizing silk. Such saltstypically create high ionic strength in the aqueous solutions whichdestabilizes the beta-sheet interactions in silk fibroin. In someembodiments, a combination of chaotropic agents is used to facilitatethe dissolution of silk fibroin. In some embodiments, a chaotropic agentis used to dissolve raw silk during processing.

Molecular Weight

In some embodiments, processed silk preparations are characterized bythe molecular weight of proteins present in the preparations. Differentmolecular weights may be present as a result of different levels of silkfibroin dissociation and/or fragmentation during degumming or otherprocessing. When referring to silk fibroin molecular weight herein, itshould be understood that the molecular weight may be associated withsilk fibroin polymers, silk fibroin monomers, silk fibroin heavy and/orlight chains, silk fibroin fragments, or variants, derivates, ormixtures thereof. Accordingly, silk fibroin molecular weight values mayvary depending on the nature of the silk fibroin or silk fibroinpreparation. In some embodiments, processed silk preparations arecharacterized by average molecular weight of silk fibroin fragmentspresent in the preparation; by a range of silk fibroin fragmentmolecular weights; by a threshold of silk fibroin fragment molecularweights; or by combinations of averages, ranges, and thresholds.

In some embodiments, processed silk preparation may include silk fibroinwith a molecular weight of, average molecular weight of, upper molecularweight threshold of, lower molecular weight threshold of, or range ofmolecular weights with an upper or lower range value of from about 1 kDato about 4 kDa, from about 2 kDa to about 5 kDa, from about 3.5 kDa toabout 10 kDa, from about 5 kDa to about 20 kDa, from about 7.5 kDa toabout 32.5 kDa, from about 7.5 kDa to about 50 kDa, from about 7.5 kDato about 100 kDa, from about 7.5 kDa to about 150 kDa, from about 7.5kDa to about 200 kDa, from about 7.5 kDa to about 250 kDa, from about 10kDa to about 35 kDa, from about 15 kDa to about 40 kDa, from about 20kDa to about 45 kDa, from about 25 kDa to about 50 kDa, from about 30kDa to about 55 kDa, from about 35 kDa to about 60 kDa, from about 40kDa to about 65 kDa, from about 45 kDa to about 70 kDa, from about 50kDa to about 75 kDa, from about 55 kDa to about 80 kDa, from about 60kDa to about 85 kDa, from about 65 kDa to about 90 kDa, from about 70kDa to about 95 kDa, from about 75 kDa to about 100 kDa, from about 80kDa to about 105 kDa, from about 85 kDa to about 110 kDa, from about 90kDa to about 115 kDa, from about 95 kDa to about 120 kDa, from about 100kDa to about 125 kDa, from about 105 kDa to about 130 kDa, from about110 kDa to about 135 kDa, from about 115 kDa to about 140 kDa, fromabout 120 kDa to about 145 kDa, from about 125 kDa to about 150 kDa,from about 130 kDa to about 155 kDa, from about 135 kDa to about 160kDa, from about 140 kDa to about 165 kDa, from about 145 kDa to about170 kDa, from about 150 kDa to about 175 kDa, from about 160 kDa toabout 200 kDa, from about 170 kDa to about 210 kDa, from about 180 kDato about 220 kDa, from about 190 kDa to about 230 kDa, from about 200kDa to about 240 kDa, from about 210 kDa to about 250 kDa, from about220 kDa to about 260 kDa, from about 230 kDa to about 270 kDa, fromabout 240 kDa to about 280 kDa, from about 250 kDa to about 290 kDa,from about 260 kDa to about 300 kDa, from about 270 kDa to about 310kDa, from about 280 kDa to about 320 kDa, from about 290 kDa to about330 kDa, from about 300 kDa to about 340 kDa, from about 310 kDa toabout 350 kDa, from about 320 kDa to about 360 kDa, from about 330 kDato about 370 kDa, from about 340 kDa to about 380 kDa, from about 350kDa to about 390 kDa, from about 360 kDa to about 400 kDa, from about370 kDa to about 410 kDa, from about 380 kDa to about 420 kDa, fromabout 390 kDa to about 430 kDa, from about 400 kDa to about 440 kDa,from about 410 kDa to about 450 kDa, from about 420 kDa to about 460kDa, from about 430 kDa to about 470 kDa, from about 440 kDa to about480 kDa, from about 450 kDa to about 490 kDa, from about 460 kDa toabout 500 kDa, or greater than 500 kDa.

In one embodiment, the silk preparation may include silk fibroin with amolecular weight of or an average molecular weight of 5-60 kDa.

In one embodiment, the silk preparation may include silk fibroin with amolecular weight of or an average molecular weight of 30-60 kDa. In oneaspect, silk fibroin in this range maybe referred to as low molecularweight.

In one embodiment, the silk preparation may include silk fibroin with amolecular weight of or an average molecular weight of 100-300 kDa. Inone aspect, silk fibroin in this range maybe referred to as highmolecular weight.

In one embodiment, the silk preparation may include silk fibroin with amolecular weight of or an average molecular weight of 361 kDa.

Processed silk preparations may be analyzed, for example, bypolyacrylamide gel electrophoresis (PAGE) alongside molecular weightstandards to determine predominate molecular weights of proteins and/orpolymers present. Additional methods for determining the molecularweight range or average molecular weight for a processed silkpreparation may include, but are not limited to, sodium dodecyl sulfate(SDS)-PAGE, size-exclusion chromatography (SEC), high pressure liquidchromatography (HPLC), non-denaturing PAGE, and mass spectrometry (MS).

Processed silk preparations may include low molecular weight silkfibroin. As used herein, the term “low molecular weight silk fibroin”refers to silk fibroin with a molecular weight below 200 kDa. Someprocessed silk preparations may include high molecular weight silkfibroin. As used herein, the term “high molecular weight silk fibroin”refers to silk fibroin with a molecular weight equal to or greater than200 kDa. In some embodiments, the silk fibroin molecular weight isdefined by the degumming boiling time. In some embodiments, silk fibroinwith a 480-minute boil, or “mb” is considered to be low molecular weightsilk fibroin. In some embodiments, silk fibroin with a 120-minute boil,or “mb” is considered to be high molecular weight silk fibroin.

In some embodiments, silk fibroin molecular weight is modulated by themethod of degumming used during processing. In some embodiments, longerheating times during degumming are used (e.g., see InternationalPublication No. WO2014145002, the contents of which are hereinincorporated by reference in their entirety). Longer heating (e.g.,boiling) time may be used during the degumming process to prepare silkfibroin with lower average molecular weights. In some embodiments,heating times may be from about 1 min to about 5 min, from about 2 minto about 10 min, from about 5 min to about 15 min, from about 10 min toabout 25 min, from about 20 min to about 35 min, from about 30 min toabout 50 min, from about 45 min to about 75 min, from about 60 min toabout 95 min, from about 90 min to about 125 min, from about 120 min toabout 175 min, from about 150 min to about 200 min, from about 180 minto about 250 min, from about 210 min to about 350 min, form about 240min to about 400 min, from about 270 min to about 450 min, from about300 min to about 480 min, or more than 480 min. Additionally, the sodiumcarbonate concentration used in the degumming process, as well as theheating temperature, may also be altered to modulate the molecularweight of silk fibroin. In one embodiment, the alteration may cause anincrease in the molecular weight of silk fibroin. As compared to silkfibroin where the sodium carbonate concentration and/or the heatingtemperature was not altered, the increase of the molecular weight may be1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or greaterthan 99% higher. In one embodiment, the alteration may cause a decreasein the molecular weight of silk fibroin. As compared to silk fibroinwhere the sodium carbonate concentration and/or the heating temperaturewas not altered, the decrease of the molecular weight may be 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or greater than 99%lower.

In some embodiments, silk fibroin molecular weight may be presumed,without actual analysis, based on methods used to prepare the silkfibroin. For example, silk fibroin may be presumed to be low molecularweight silk fibroin or high molecular weight silk fibroin based on thelength of time that heating is carried out (e.g., by minute boil value).

In some embodiments, SBPs include a plurality of silk fibroin fragmentsgenerated using a dissociation procedure. The dissociation procedure mayinclude one or more of heating, acid treatment, chaotropic agenttreatment, sonication, and electrolysis. Some SBPs include a pluralityof silk fibroin fragments dissociated from raw silk, silk fiber, and/orsilk fibroin by heating. The heating may be carried out at a temperatureof from about 30° C. to about 1,000° C. In some embodiments, heating iscarried out by boiling. The raw silk, silk fiber, and/or silk fibroinmay be boiled for from about 1 second to about 24 hours.

Silk Fibroin Boiling Time

SBP formulations with processed silk with varying molecular weights. Insome embodiments, the silk fibroin molecular weight is defined by thedegumming boiling time. In some embodiments, silk fibroin with a480-minute boil, or “mb” may produce be a low molecular weight silkfibroin when compared to a silk fibroin produced with a 120-minute boil,or “mb”. In some aspects, the 120 mb silk fibroin is considered to behigh molecular weight silk fibroin in comparison to the 480 mb silkfibroin. In some embodiments, a longer boiling time is considered to belower molecular weight silk fibroin. In some embodiments, a shorterboiling time is considered to be a higher molecular weight silk fibroin.In some embodiments, the boiling time is about 15 minutes, about 30minutes, about 60 minutes, about 90 minutes, about 120 minutes, or about480 minutes. In some embodiments, an SBP is prepared with processed silkwith a single boiling time. In some embodiments, an SBP contains a blendof processed silk with different boiling times.

In one embodiment, the SBP formulation includes 30 mb silk fibroin.

In one embodiment, the SBP formulation includes 60 mb silk fibroin.

In one embodiment, the SBP formulation includes 90 mb silk fibroin.

In one embodiment, the SBP formulation includes 120 mb silk fibroin.

In one embodiment, the SBP formulation includes 480 mb silk fibroin.

Purification and Concentration

In some embodiments, processed silk preparations may be purified.Purification, as used herein, refers to any process used to segregate orextract one entity from another. In some embodiments, purification ismanual or automated. Purification may include the removal of salts,impurities, or contaminants from processed silk preparations.

In some embodiments, processed silk may be purified by concentrationfrom a processed silk solution. Methods of concentrating silk fibroinfrom processed silk solutions may include any of those described in theInternational Publication No. WO2017139684, the contents of which areincorporated herein by reference in their entirety. In some embodiments,purification and/or concentration may be carried out by one or more ofdialysis, centrifugation, air drying, vacuum drying, filtration, and/orTangential Flow Filtration (TFF).

In some embodiments, processed silk solutions may be purified bydialysis. Dialysis may be carried out to remove undesired salts and/orcontaminants. In some embodiments, processed silk solutions areconcentrated via dialysis. Purification and/or concentration ofprocessed silk by dialysis may be carried out as described inInternational Publication No. WO2005012606, the contents of which areherein incorporated by reference in their entirety. In some embodiments,the dialysis is performed against a hygroscopic polymer to concentratethe silk fibroin solution. In some embodiments the dialysis is manual,with the use of a membrane and manual solvent changes. In someembodiments, the solvent is changed between 1 and 10 times over thecourse of the procedure. In some embodiments, the membrane is a dialysiscassette. The dialysis cassette may be a slide-a-lyzer dialysiscassette. In some embodiments, the membrane is dialysis tubing. Thedialysis tubing may be regenerated cellulose dialysis tubing and/orsnake skin. The dialysis tubing or cassette may be rinsed in distilledwater for 30 minutes to prepare the membrane for use. In someembodiments, the dialysis tubing has a molecular weight cutoff of 3.5kDa. In some embodiments, the dialysis is performed at a temperature offrom about 1° C. to about 30° C. In some embodiments, dialysis isperformed at room temperature. In other embodiments, the dialysis isperformed at 4° C. Dialysis may be performed until desiredconcentrations of silk fibroin and salt are obtained from processed silksolutions. Dialysis may be performed for periods of time from about 30minutes to about 24 hours or beyond. For example, dialysis may becarried out for from about 30 minutes to about 2 hours, from about 1hour to about 6 hours, from about 3 hours to about 10 hours, from about5 hours, to about 12 hours, from about 7 hours to about 15 hours, fromabout 11 hours to about 20 hours, or from about 16 hours to about 24hours.

In some embodiments, dialysis may be automated. The dialysis may use anautomated water change system. Such systems may include tanks of up to10 L and may be able to hold multiple dialysis cassettes (e.g., seeInternational Publication No. WO2017106631, the contents of which areherein incorporated by reference in their entirety). Automated equipmentmay enable purification of larger volumes of solution with greaterefficiency. Automated controllers, programmed with the proper times andvolumes, may be used to facilitate changes of solvent or buffer over thecourse of dialysis. The solvent may be replaced from about 1 to about 20times or more during dialysis. In some embodiments, automated dialysismay be completed in about 48 hours.

Dialysis may be performed with various solvents depending on the natureof the preparation being processed. In some embodiments the solvent maybe water. In some embodiments, the solvent may be an aqueous solution.In some embodiments the solvent includes a hygroscopic polymer.Hygroscopic polymers may include, but are not limited to polyethyleneglycol (PEG), polyethylene oxide (PEO), collagen, fibronectin, keratin,polyaspartic acid, polylysine, alginate, chitosan, chitin, hyaluronicacid, pectin, polycaprolactone, polylactic acid, polyglycolic acid,polyhydroxyalkanoates, dextrans, and polyanhydrides. Additional examplesof hygroscopic polymers and related dialysis methods that may beemployed include any of those found in International Publication NumbersWO2005012606, WO2005012606 and WO2017106631, and U.S. Pat. Nos.6,302,848, 6,395,734, 6,127,143, 5,263,992, 6,379,690, 5,015,476,4,806,355, 6,372,244, 6,310,188, 5,093,489, 6,325,810, 6,337,198,6,267,776, 5,576,881, 6,245,537, 5,902,800, and 5,270,419, the contentsof each of which are herein incorporated by reference in their entirety.Hygroscopic polymer concentrations may be from about 20% (w/v) to about50% (w/v). In some embodiments, dialysis may be performed in a stepwisemanner in a urea solution, and the urea solution may be subsequently bereplaced with urea solutions of a lower concentration during bufferchanges, until it is ultimately replaced with water, as described inZheng et al. (2016) Journal of Biomaterials Applications 31:450-463.

In some embodiments, processed silk preparations may be purified byfiltration. Such filtration may include trans flow filtration (TFF),also known as tangential flow filtration. During TFF, solutions may bepassed across a filter membrane. Anything larger than the membrane poreswould is retained, and anything smaller passes through the membrane(e.g., see International Publication No. WO2017106631, the contents ofwhich are herein incorporated by reference in their entirety). With thepositive pressure and flow along the membrane, instead of through it,particles trapped in the membrane may be washed away. TFF may be carriedout using an instrument. The instrument may be automated. The membranesmay be housed in TFF tubes with vertical inlets and outlets. The flow ofsolvent may be controlled by peristaltic pumps. Some TFF tubes mayinclude a dual chamber element. The dual chamber element may enable TFFfiltration of processed silk solutions at higher concentrations, whilereducing aggregation via the reduction of shear forces.

In some embodiments, processed silk solutions are purified and/orconcentrated by centrifugation. Centrifugation may be performed beforeor after other forms of purification, which include, but are not limitedto dialysis and tangential flow filtration. Centrifugation times andspeeds may be varied to optimize purification and/or concentrationaccording to optimal time frames. Purification and/or concentration bycentrifugation may include pelleting of the processed silk and removalof supernatant. In some cases, centrifugation is used to push solventthrough a filter, while retaining processed silk. Centrifugation may berepeated as many times as needed. In some embodiments, silk fibroinsolutions are centrifuged two or more times during concentration and/orpurification.

Drying Methods

In some embodiments, processed silk preparations may be dried to removesolvent. In some embodiments, SBP formulations may be rinsed prior todrying. Methods of drying may include, but are not limited to, airdrying, oven drying, lyophilization, spray drying, spray freezing, andvacuum drying. Drying may be carried out to alter the consistency and/orother properties of processed silk preparations. One or more compoundsor excipients may be combined with processed silk preparations toimprove processed silk recovery and/or reconstitution after the dryingprocess. For example, sucrose may be added to improve silk fibroinrecovery and reconstitution from dried solutions. In some embodiments,drying may be carried out in the fabrication of a processed silk formator a SBP. Examples include, but are not limited to fabrication offibers, nanofibers, mats, films, foams, membranes, rods, tubes, gels,hydrogels, microspheres, nanospheres, solutions, patches, grafts andpowders. In some embodiments, drying processed silk may be carried outby oven drying, lyophilizing, and/or air drying.

Oven drying refers to any drying method that uses an oven. According tosome methods, ovens are maintained at temperatures of from about 30° C.to about 90° C. or more. In some embodiment, oven drying is carried outat a temperature of 60° C. Processed silk preparations may be placed inovens for a period of from about 1 hour to about 24 hours or more. Inone embodiment, SBP formulations are oven dried at 60° C. for 2 hours.Oven drying may be used to dry silk fibroin preparations. In someembodiments, silk fibroin preparations are oven dried for 16 hours at60° C. to obtain a desired format. In some cases, silk fibroin solutionsare oven dried overnight. Examples of formats obtained by oven dryingmay include, but are not limited to, fibers, nanofibers, mats, films,foams, membranes, rods, tubes, gels, hydrogels, microspheres,nanospheres, solutions, patches, grafts, and powders.

In some embodiments, processed silk preparations are freeze dried.Freeze drying may be carried out by lyophilization. Freeze drying mayrequire processed silk preparations to be frozen prior to freeze drying.Freezing may be carried out at temperatures of from about 5° C. andabout −85° C. In some embodiments, freeze drying is carried out bylyophilization for up to 75 hours. In some embodiments, lyophilizationis used to prepare processed silk formats or SBPs. Such formats mayinclude, but are not limited to, fibers, nanofibers, mats, films, foams,membranes, rods, tubes, gels, hydrogels, microspheres, nanospheres,solutions, patches, grafts and powders. The use of lyophilization tofabricate SBPs may be carried out according to any of the methodsdescribed in Zhou et al. (2017) Acta Biomater S1742-7061(17)30569; Yanget al. (2017) Int J Nanomedicine 12:6721-6733; Seo et al. (2017) JBiomater Appl 32(4):484-491; Ruan et al. (2017) Biomed Pharmacother97:600-606; Wu et al. (2017) J Mech Behav Biomed Mater 77:671-682; Zhaoet al. (2017) Materials Letters 211:110-113; Chen et al (2017) PLoS One12(1l):e0187880; Min et al. (2017) Int J Biol Macromol 17: 32855-8; Sunet al. Journal of Materials Chemistry B 5:8770; and Thai et al. J BiomedMater (2017) 13(1):015009, the contents of each of which are hereinincorporated by reference in their entirety.

In some embodiments, processed silk preparations may be dried by airdrying. “Air drying,” as used herein refers to the removal of moistureby exposure to ambient or circulated gasses. Air drying may includeexposing a preparation to air at room temperature (from about 18° C. toabout 29° C.). Air drying may be carried out for from about 30 minutesto about 24 hours or more. In some embodiments, silk fibroinpreparations are air dried to prepare SBPs. SBP formats that may beprepared may include, but are not limited to, fibers, nanofibers, mats,films, foams, membranes, rods, tubes, gels, hydrogels, microspheres,nanospheres, solutions, patches, grafts and powders. Some examples ofthe use of air drying for fabrication of SBPs are presented in Susaninet al. (2017) Fibre Chemistry 49(2):88-96; Lo et al. J Tissue Eng RegenMed (2017) doi.10.1002/term.2616; and Mane et al. Scientific Reports7:15531, the contents of each of which are herein incorporated byreference in their entirety.

Spinning

In some embodiments, processed silk may be prepared by spinning. As usedherein, the term “spinning” refers to a process of twisting materialstogether. Spinning may include the process of preparing a silk fiber bytwisting silk proteins as they are secreted from silk producers. Otherforms of spinning include spinning one or more forms of processed silktogether to form a thread, filament, fiber, or yarn. The processed silkmay already consist of a filamentous format prior to spinning. In someembodiments, processed silk is processed by spinning from anon-filamentous format (e.g., from a film, mat, or solution).

In some embodiments, spinning includes the technique of electrospinning.Electrospinning may be used to prepare silk fibers from silk fibroin.The silk fibroin may be dissolved in water or an aqueous solution beforeelectrospinning. In other embodiments, silk fibroin is dissolved in anorganic solvent before electrospinning. The organic solvent may behexafluoroisopropanol (HFIP). In some embodiments, electrospinning maybe carried out as described in Yu et al. (2017) Biomed Mater Res A doi.10.1002/jbm.a.36297 or Chantawong et al. (2017) Mater Sci Mater Med28(12):191, the contents of each of which are herein incorporated byreference in their entirety.

Electrospinning typically includes the use of an electrospinningapparatus. Processed silk may be added to the apparatus to produce silkfiber. The processed silk may be silk fibroin in solution.Electrospinning apparatus components may include one or more of aspinneret (also referred to as a spinnerette), needle, mandrel, powersource, pump, and grounded collector. The apparatus may apply voltage tothe dissolved silk fibroin, causing electrostatic repulsion thatgenerates a charged liquid that is extruded from the end. Electrostaticrepulsion also enables fiber elongation as it forms, and charged liquidcohesion prevents it from breaking apart. Resulting fiber may bedeposited on the collector. In some embodiments, electrospinning methodsmay be carried out according to those described in European Patent No.EP3206725; Manchineella et al. (2017) European Journal of OrganicChemistry 30:4363-4369; Park et a. (2017) Int J BiomacromolS0141-8130(17):32645-4; Wang et al. (2017) J Biomed Mater Res Adoi.10.1002/jbm.a.36225; Chendang et al. (2017) J Biomaterials andTissue Engineering 7:858-862; Kambe et al. (2017) Materials (Basel)10(10):E1153; Chouhan et al. (2017) J Tissue Eng Reneg Meddoi.10.1002/term.2581; Genovese et al. (2017) ACS Appl Mater Interfacesdoi.10.1021acsami.7b13372; Yu et al. (2017) Biomed Mater Res A doi.10.1002/jbm.a.36297; Chantawong et al. (2017) Mater Sci Mater Med28(12):191, the contents of each of which are herein incorporated byreference in their entirety.

In some embodiments, spinning may be carried out as dry spinning. Dryspinning may be carried out using a dry spinning apparatus. Dry spinningmay be used to prepare silk fibers from processed silk preparations. Thepreparations may include silk fibroin solutions. The preparations may beaqueous solutions. Dry spinning apparatuses typically use hot air to dryprocessed silk as it is extruded. In some embodiments, dry spinning maybe carried out according to any of the methods presented in Zhang et al.(2017) Int J Biol Macromol pii:S0141-8130(17):32857, the contents ofwhich are herein incorporated by reference in their entirety.

Spraying

In some embodiments, processing methods include spraying. As usedherein, the term “spraying” refers to the sprinkling or showering of acompound or composition in the form of small drops or particles.Spraying may be used to prepare SBPs by spraying processed silk.Spraying may be carried out using electrospraying. Processed silk usedfor spraying may include processed silk in solution. The solution may bea silk fibroin solution. Solutions may be aqueous solutions. Somesolutions may include organic solvents. Electrospraying may be carriedout in a manner similar to that of electrospinning, except that thecharged liquid lacks cohesive force necessary to prevent extrudingmaterial from breaking apart. In some embodiments, spraying methods mayinclude any of those presented in United States Publication No.US2017/333351 or Cao et al. (2017) Scientific Reports 7:11913, thecontents of each of which are herein incorporated by reference in theirentirety. In some embodiments, electrospray methods include a coaxialsystem for coaxial spraying.

Precipitation

In some embodiments, processing methods include precipitation. As usedherein, the term “precipitation” refers to the deposition of a substancein solid form from a solution. Precipitation may be used to obtain solidprocessed silk from processed silk solutions. The processed silk may besilk fibroin. Processed silk may be precipitate from a solution. Thesolvent may be aqueous. In some embodiments, the solvent is organic.Examples of organic solvents include, but are not limited to. HFIP,methanol, ethanol, and other alcohols. In some embodiments, the solventis water. In some embodiments the solvent is a mixture of an organicsolvent and water. Aqueous solvents may contain one or more salts.Processed silk may be precipitated from processed silk solutions bymodulating one or more components of the solution to alter thesolubility of the processed silk and promote precipitation. Additionalprocessing steps may be employed to initiate or speed precipitation.Such methods may include, but are not limited to sonication,centrifugation, increasing the concentration of processed silk, alteringthe concentration of salt, adding additional salt or salts, altering thepH, applying shear stress, adding excipients, or applying chemicalmodifications.

Altering Mechanical Properties

In some embodiments, the mechanical properties of processed silk may bealtered by modulating physical and/or chemical properties of theprocessed silk. The mechanical properties include, but are not limitedto, mechanical strength, tensile strength, elongation capabilities,elasticity, compressive strength, stiffness, shear strength, toughness,torsional stability, temperature stability, moisture stability,viscosity, and reeling rate. Examples of the physical and chemicalproperties used to tune the mechanical properties of processed silkinclude, but are not limited to, the temperature, formulations, silkconcentration, β-sheet content, crosslinking, the molecular weight ofthe silk, the storage of the silk, storage, methods of preparation,dryness, methods of drying, purity, and degumming. Methods of tuning themechanical strength of processed silk are taught in International PatentApplication Publication No. WO2017123383, European Patent No. EP2904134,European Patent No. EP3212246, Fang et al., Wu et al., Susanin et al.,Zhang et al., Jiang et al. Yu et al., Chantawong et al., and Zhang etal. (Fang et al. (2017) Journal of Materials Chemistry B5(30):6042-6048.; Wu et al. (2017) J Mech Behav Biomed Mater77:671-682.; Susanin et al. (2017) Fibre Chemistry 49(2):88-96.; Zhanget al. (2017) Fibers and Polymers 203:9-16.; Jiang et al. (2017) JBiomater Sci Polym Ed 15:1-36; Yu et al. (2017) Biomed Mater Res A doi.10.1002/jbm.a.36297.; Chantawong et al. (2017) Mater Sci Mater Med28(12):191.; Zhang et al. (2017) Int J BiomacromolS0141-8310(17):32857), the contents of each of which are hereinincorporated by reference in their entirety.

In some embodiments, the excipients which may be incorporated in aformulation may be used to control the modulus of processed silkpreparations. In some embodiments, these processed silk preparations arehydrogels. In some embodiments, processed silk hydrogels are preparedwith different excipients and tested for their mechanical properties,including the modulus. Processed silk preparations may be assessed formodulus, shear storage modulus, shear loss modulus, phase angle, andviscosity using a rheometer, and/or any other method known to oneskilled in the art. Processed silk preparations may be tested bothbefore and after gelation. In some embodiments, processed silkpreparations are prepared, optionally with different excipients, andtested for their mechanical properties, including the modulus, shearstorage modulus, the shear loss modulus, phase angle, and viscosity. Asused herein, the term “shear storage modulus” refers to the measure of amaterial's elasticity or reversible deformation as determined by thematerial's stored energy. As used herein, the term “shear loss modulus”refer to the measure of a material's ability to dissipate energy,usually in the form of heat. As used herein, the term “phase angle”refers to the difference in the stress and strain applied to a materialduring the application of oscillating shear stress. As used herein, theterm “viscosity” refers to a material's ability to resist deformationdue to shear forces, and the ability of a fluid to resist flow. In someembodiments, processed silk hydrogels may possess similar viscositiesbut vary in the modulus.

In some embodiments, the processed silk preparations may shear thin ordisplay shear thinning properties. As used herein, the term “shearthinning” refers to a decrease in viscosity at increasing shear rates.As used herein, the term “shear rate” refers to the rate of change inthe ratio of displacement of material upon the application of a shearforce to the height of the material. This ratio is also known as strain.In some embodiments, the concentration of processed silk may enable silkpreparations to shear thin. In some embodiments the silk preparation isan SBP. In some embodiments, the SBP is a hydrogel. In some embodiments,the molecular weight of processed silk hydrogels may enable hydrogels toshear thin. In some embodiments, hydrogels prepared with low molecularweight silk fibroin may be injected with much less force than hydrogelsof similar viscosity that are prepared with higher molecular weight silkfibroin. In some embodiments, hydrogels with low molecular weight silkfibroin display higher viscosity than hydrogels with high molecularweight silk fibroin.

Modulating Degradation/Resorption

In some embodiments, processed silks are or are processed to bebiocompatible. As used herein, a “biocompatible” substance is anysubstance that is not harmful to most living organisms or tissues. Withsome processed silk, degradation may result in products that arebiocompatible, making such processed silk attractive for a variety ofapplications. Some processed silk may degrade into smaller proteins oramino acids. Some processed silk may be resorbable under physiologicalconditions. In some embodiments, products of silk degradation may beresorbable in vivo. In some embodiments, the rate of degradation ofprocessed silk may be tuned by altering processed silk properties.Examples of these properties include, but are not limited to, type andconcentration of certain proteins, P-sheet content, crosslinking, silkfibroin molecular weight, and purity. In some embodiments, rate ofprocessed silk degradation may be modulated by method of storage,methods of preparation, dryness, methods of drying, reeling rate, anddegumming process.

In some embodiments, the bioresorbability and degradation of processedsilk is modulated by the addition of sucrose, as taught in Li et al. (Liet al. (2017) Biomacromolecules 18(9):2900-2905), the contents of whichare herein incorporated by reference in their entirety. Processed silkmay be formulated with sucrose to enhance thermal stability.Furthermore, processed silk with sucrose may also be formulated withantiplasticizing agents to further enhance thermal stability ofprocessed silk, SBPs, and/or therapeutic agents included in SBPs.Methods of increasing thermal stability using antiplasticizing agentsmay include any of those described in Li et al. (Li et al. (2017)Biomacromolecules 18(9):2900-2905), the contents of which are hereinincorporated by reference in their entirety. In some embodiments, theaddition of sucrose to processed silk preparations prior tolyophilization leads to an increased reconstitution efficiency. In someembodiments, the addition of sucrose may be used to create highermolecular weight processed silk preparations as well as to maintain longterm storage stability. In some embodiments, the incorporation ofsucrose into processed silk preparations described herein enables slowerfreezing during lyophilization cycle.

In some embodiments, the bioresorbability and degradation of processedsilk may be tuned through formulation with additional bioresorbablepolymer matrices, as taught in International Publication NumbersWO2017177281 and WO2017179069, the contents of each of which are hereinincorporated by reference in their entirety. In some embodiments, thepolymer matrix is polyurethane. In some embodiments, these polymermatrices may be polycaprolactone and a ceramic filler. The ceramicfiller may include MgO.

In some embodiments, the bioresorbability and degradation of processedsilk is tuned through the fabrication of a composite scaffold. Compositescaffolds, combinations of scaffolds or scaffolds formed form more thanone material, may be formed from two or more processed silkpreparations. In some embodiments, processed silk scaffolds comprising acombination of silk fibroin microspheres within a larger processed silkpreparation may demonstrate slower degradation in comparison with otherscaffolds, as taught in European Patent No. EP3242967, the contents ofwhich are herein incorporated by reference in their entirety.

Excipients

In some embodiments, SBPs include one or more excipients. As usedherein, the term “excipient” refers to any substance included in acomposition with an active agent or primary component, often serving asa carrier, diluent, or vehicle for the active agent or primarycomponent. In some embodiments, excipients may be compounds orcompositions approved for use by the US Food and Drug Administration(FDA). In some embodiments, SBPs may include excipients that increaseSBP stability or stability of one or more other SBP components. SomeSBPs may include an excipient that modulates payload release. Excipientsmay include, but are not limited to, solvents, diluents, liquidvehicles, dispersion or suspension media or aids, surfactants,thickening agents, emulsifying agents, lipids, liposomes, isotonicagents, buffers, and preservatives. In some embodiments, excipientsinclude lipidoids, lipid nanoparticles, polymers, lipoplexes, particles,core-shell nanoparticles, peptides, proteins, cells, hyaluronidase,and/or nanoparticle mimics. In some embodiments, processed silk and/orSBPs may be used as an excipient.

In some embodiments, excipients included in SBPs may be selected fromone or more of lactose, phosphate salts, sodium chloride, potassiumphosphate monobasic, potassium phosphate dibasic, sodium phosphatedibasic, sodium phosphate monobasic, polysorbate 80, phosphate buffer,phosphate buffered saline, sodium hydroxide, sorbitol, sucrose,mannitol, lactose USP, Starch 1500, microcrystalline cellulose, Avicel,dibasic calcium phosphate dehydrate, tartaric acid, citric acid, fumaricacid, succinic acid, malic acid, hydrochloric acid,polyvinylpyrrolidone, copolymers of vinylpyrrolidone and vinylacetate,hydroxypropylcellulose, hydroxyethylcellulose,hydroxypropylmethylcellulose, polyvinyl alcohol, polyethylene glycol,acacia, and sodium carboxymethylcellulose. Excipients may includesucrose. Excipients may be present in SBPs at any concentration. In someembodiments, excipients are present at a concentration of from about0.0001% weight per weight (w/w) of excipient to total SBP weight toabout 20% (w/w). In some embodiments, excipients are present at aconcentration of from about 1% (w/w) to about 20% (w/w).

In some embodiments, excipients included in SBPs may be selected fromone or more of sorbitol, triethylamine, 2-pyrrolidone,alpha-cyclodextrin, benzyl alcohol, beta-cyclodextrin, dimethylsulfoxide, dimethylacetamide (DMA), dimethylformamide, ethanol,gamma-cyclodextrin, glycerol, glycerol formal, hydroxypropylbeta-cyclodextrin, kolliphor 124, kolliphor 181, kolliphor 188,kolliphor 407, kolliphor EL (cremaphor EL), cremaphor RH 40, cremophorRH 60, dalpha-tocopherol, PEG 1000 succinate, polysorbate 20,polysorbate 80, solutol HS 15, sorbitan monooleate, poloxamer-407,poloxamer-188, Labrafil M-1944CS, Labrafil M-2125CS, Labrasol, Gellucire44/14, Softigen 767, mono- and di-fatty acid esters of PEG 300, PEG 400,or PEG 1750, kolliphor RH60, N-methyl-2-pyrrolidone, castor oil, cornoil, cottonseed oil, olive oil, peanut oil, peppermint oil, saffloweroil, sesame oil, soybean oil, hydrogenated vegetable oils, hydrogenatedsoybean oil, medium chain triglycerides of coconut oil, medium chaintriglycerides of palm seed oil, beeswax, d-alpha-tocopherol, oleic acid,medium-chain mono-glycerides, medium-chain di-glycerides,alpha-cyclodextrin, betacyclodextrin, hydroxypropyl-beta-cyclodextrin,sulfo-butylether-beta-cyclodextrin, hydrogenated soyphosphatidylcholine, distearoylphosphatidylglycerol,L-alphadimyristoylphosphatidylcholine,L-alpha-dimyristoylphosphatidylglycerol, PEG 300, PEG 300caprylic/capric glycerides (Softigen 767), PEG 300 linoleic glycerides(Labrafil M-2125CS), PEG 300 oleic glycerides (Labrafil M-1944CS), PEG400, PEG 400 caprylic/capric glycerides (Labrasol), polyoxyl 40 stearate(PEG 1750 monosterate), polyoxyl 8 stearate (PEG 400 monosterate),polysorbate 20, polysorbate 80, polyvinyl pyrrolidone, propylenecarbonate, propylene glycol, solutol HS15, sorbitan monooleate (Span20), sulfobutylether-beta-cyclodextrin, transcutol, triacetin,1-dodecylazacyclo-heptan-2-one, caprolactam, castor oil, cottonseed oil,ethyl acetate, medium chain triglycerides, methyl acetate, oleic acid,safflower oil, sesame oil, soybean oil, tetrahydrofuran, glycerin, andPEG 4 kDa. Such SBPs may include hydrogels. In some embodiments, SBPhydrogels include one or more of polysorbate 80, poloxamer-188, PEG 4kDa, and glycerol.

In one embodiment, the excipient is sorbitol.

In one embodiment, the excipient is mannitol.

Gelling Agents

In some embodiments, excipients may include gelling agents. As usedherein, the term “gelling agent” refers to any substance that promotesviscosity and/or polymer cross-linking in compositions. Non-limitingexamples of gelling agents include glycerol, glycerophosphate, sorbitol,hydroxyethyl cellulose, carboxymethyl cellulose, triethylamine,triethanolamine, 2-pyrrolidone, alpha-cyclodextrin, benzyl alcohol,beta-cyclodextrin, dimethyl sulfoxide, dimethylacetamide (DMA),dimethylformamide, ethanol, gamma-cyclodextrin, glycerol formal,hydroxypropyl beta-cyclodextrin, kolliphor 124, kolliphor 181, kolliphor188, kolliphor 407, kolliphor EL (cremaphor EL), cremaphor RH 40,cremaphor RH 60, d-alpha-tocopherol, PEG 1000 succinate, polysorbate 20,polysorbate 80, solutol HS 15, sorbitan monooleate, poloxamer-407,poloxamer-188, Labrafil M-1944CS, Labrafil M-2125CS, Labrasol, Gellucire44/14, Softigen 767, mono- and di-fatty acid esters of PEG 300, PEG 400,PEG 4 kDa, or PEG 1750, kolliphor RH60, N-methyl-2-pyrrolidone, castoroil, corn oil, cottonseed oil, olive oil, peanut oil, peppermint oil,safflower oil, sesame oil, soybean oil, hydrogenated vegetable oils,hydrogenated soybean oil, and medium-chain triglycerides of coconut oiland palm seed oil, beeswax, d-alpha-tocopherol, oleic acid, medium-chainmono- and diglycerides, alpha-cyclodextrin, beta-cyclodextrin,hydroxypropyl-beta-cyclodextrin, sulfo-butylether-beta-cyclodextrin,hydrogenated soy phosphatidylcholine, distearoylphosphatidylglycerol,L-alpha-dimyristoylphosphatidylcholine,L-alphadimyristoylphosphatidylglycerol, PEG 300, PEG 300 caprylic/capricglycerides (Softigen 767), PEG 300 linoleic glycerides (LabrafilM-2125CS), PEG 300 oleic glycerides (Labrafil M-1944CS), PEG 400, PEG400 caprylic/capric glycerides (Labrasol), polyoxyl 40 stearate (PEG1750 monosterate), polyoxyl 8 stearate (PEG 400 monosterate),polysorbate 20, polysorbate-SO, polyvinyl pyrrolidone, polyvinylpyrrolidone-12, polyvinyl pyrrolidone-17, propylene carbonate, propyleneglycol, solutol HS 15, sorbitan monooleate (Span 20),sulfobutylether-beta-cyclodextrin, transcutol, triacetin,1-dodecylazacyclo-heptan-2-one, caprolactam, castor oil, cottonseed oil,ethyl acetate, medium chain triglycerides, methyl acetate, oleic acid,safflower oil, sesame oil, soybean oil, tetrahydrofuran, and glycerin.Additional examples of gelling agents include acacia, alginic acid,bentonite, CARBOPOLS® (also known as carbomers), carboxymethylcellulose, ethylcellulose, gelatin, hydroxy ethyl cellulose,hydroxypropyl cellulose, magnesium aluminum silicate, methylcellulose,poloxamers, polyvinyl alcohol, sodium alginate, tragacanth, and xanthangum.

PEGs which may be used as gelling agents and/or excipients may beselected from a variety of chain lengths and molecular weights. Thesecompounds are typically prepared through ethylene oxide polymerization.In some embodiments, PEGs may have a molecular weight of from about 300g/mol to about 100,000 g/mol. In some embodiments, PEGs may have amolecular weight of from about 3600 g/mol to about 4400 g/mol. In someembodiments, PEGs with a molecular weight of from about 300 g/mol toabout 3000 g/mol, from about 350 g/mol to about 3500 g/mol, from about400 g/mol to about 4000 g/mol, from about 450 g/mol to about 4500 g/mol,from about 500 g/mol to about 5000 g/mol, from about 550 g/mol to about5500 g/mol, from about 600 g/mol to about 6000 g/mol, from about 650g/mol to about 6500 g/mol, from about 700 g/mol to about 7000 g/mol,from about 750 g/mol to about 7500 g/mol, from about 800 g/mol to about8000 g/mol, from about 850 g/mol to about 8500 g/mol, from about 900g/mol to about 9000 g/mol, from about 950 g/mol to about 9500 g/mol,from about 1000 g/mol to about 10000 g/mol, from about 1100 g/mol toabout 12000 g/mol, from about 1200 g/mol to about 14000 g/mol, fromabout 1300 g/mol to about 16000 g/mol, from about 1400 g/mol to about18000 g/mol, from about 1500 g/mol to about 20000 g/mol, from about 1600g/mol to about 22000 g/mol, from about 1700 g/mol to about 24000 g/mol,from about 1800 g/mol to about 26000 g/mol, from about 1900 g/mol toabout 28000 g/mol, from about 2000 g/mol to about 30000 g/mol, fromabout 2200 g/mol to about 35000 g/mol, from about 2400 g/mol to about40000 g/mol, from about 2600 g/mol to about 45000 g/mol, from about 2800g/mol to about 50000 g/mol, from about 3000 g/mol to about 55000 g/mol,from about 10000 g/mol to about 60000 g/mol, from about 13000 g/mol toabout 65000 g/mol, from about 16000 g/mol to about 70000 g/mol, fromabout 19000 g/mol to about 75000 g/mol, from about 22000 g/mol to about80000 g/mol, from about 25000 g/mol to about 85000 g/mol, from about28000 g/mol to about 90000 g/mol, from about 31000 g/mol to about 95000g/mol, or from about 34000 g/mol to about 100000 g/mol are utilized.

Formats

SBPs may include or be prepared to conform to a variety of formats. Insome embodiments, such formats include formulations of processed silkwith various excipients and/or cargo. In some embodiments, SBP formatsinclude, but are not limited to, gels, hydrogels, implants, and rods. Insome embodiments, the formats are formulated with a therapeutic agent.

Formulations

In some embodiments, SBPs may be formulations. As used herein, the term“formulation” refers to a mixture of two or more components or theprocess of preparing such mixtures. In some embodiments, theformulations are low cost and eco-friendly. In some embodiments, thepreparation or manufacturing of formulations is low cost andeco-friendly. In some embodiments, the preparation or manufacturing offormulations is scalable. In some embodiments, SBPs are prepared byextracting silk fibroin via degumming silk yam. In some embodiments, theyam is medical grade. In some embodiments the yam may be silk sutures.The extracted silk fibroin may then be dissolved in a solvent (e.g.water, aqueous solution, organic solvent). The dissolved silk fibroinmay then be dried (e.g., oven dried, air dried, or freeze-dried). Insome embodiments, dried silk fibroin is formed into formats describedherein. In some embodiments, that format is a solution. In someembodiments, that format is a powder. In some embodiments, formulationsinclude one or more excipients, carriers, additional components, and/ortherapeutic agents to generate SBPs. In some embodiments, formulationsof processed silks are prepared during the manufacture of SBPs.

Formulation components and/or component ratios may be modulated toaffect one or more SBP properties, effects, and/or applications.Variations in the concentration of silk fibroin, choice of excipient,the concentration of excipient, the osmolarity of the formulation, andthe method of formulation represent non-limiting examples of differencesin formulation that may alter properties, effects, and applications ofSBPs. In some embodiments, the formulation of SBPs may modulate theirphysical properties. Examples of physical properties include solubility,density, and thickness. In some embodiments, the formulation of SBPs maymodulate their mechanical properties. Examples of mechanical propertiesthat may be modulated include, but are not limited to, mechanicalstrength, tensile strength, elongation capabilities, elasticity,compressive strength, stiffness, shear strength, toughness, torsionalstability, temperature stability, moisture stability, viscosity, andreeling rate.

Cargo

In some embodiments, SBPs are or include cargo. As used herein, the term“cargo” refers to any substance that is embedded in, enclosed within,attached to, or otherwise associated with a carrier. SBPs may becarriers for a large variety of cargo. Such cargo may includetherapeutic agents (e.g., biological agents, particles, lipids,liposomes, carbohydrates, small molecules, ions, metals, and minerals).In some embodiments, the cargo is or includes a payload. As used herein,the term “payload” refers to cargo that is delivered from a source orcarrier to a target. Payloads may be released from SBPs, where SBPsserve as a carrier. Where SBPs are the payload, the SBPs may be releasedfrom a source or carrier. In some embodiments, payloads remainassociated with carriers upon delivery. Payloads may be released in bulkor may be released over a period of time, also referred to herein as the“delivery period.” In some embodiments, payload release is by way ofcontrolled release. As used herein, the term “controlled release” refersto distribution of a substance from a source or carrier to a surroundingarea, wherein the distribution occurs in a manner that includes or isaffected by some manipulation, some property of the carrier, or somecarrier activity.

In some embodiments, controlled release may include a steady rate ofrelease of payload from carrier. In some embodiments, payload releasemay include an initial burst, wherein a substantial amount of payload isreleased during an initial release period followed by a period whereless payload is released. In some embodiments, release rate slows overtime. Payload release may be measured by assessing payload concentrationin a surrounding area and comparing to initial payload concentration orremaining payload concentration in a carrier or source area. Payloadrelease rate may be expressed as a quantity or mass of payload releasedover time (e.g., mg/min). Payload release rate may be expressed as apercentage of payload released from a source or carrier over a period oftime (e.g., 5%/hour). Controlled release of a payload that extends thedelivery period is referred to herein as “sustained release.” Sustainedrelease may include delivery periods that are extended over a period ofhours, days, months, or years.

Some controlled release may be mediated by interactions between payloadand carrier. Some controlled release is mediated by interactions betweenpayload or carrier with surrounding areas where payload is released.With sustained payload release, payload release may be slowed orprolonged due to interactions between payload and carrier or payload andsurrounding areas where payload is released. Payload release from SBPsmay be controlled by SBP viscosity. Where the SBP includes processedsilk gel, gel viscosity may be adjusted to modulate payload release.

In some embodiments, payload delivery periods may be from about 1 secondto about 20 seconds, from about 10 seconds to about 1 minute, from about30 seconds to about 10 minutes, from about 2 minutes to about 20minutes, from about 5 minutes to about 30 minutes, from about 15 minutesto about 1 hour, from about 45 minutes to about 2 hours, from about 90minutes to about 5 hours, from about 3 hours to about 20 hours, fromabout 10 hours to about 50 hours, from about 24 hours to about 100hours, from about 48 hours to about 2 weeks, from about 72 hours toabout 4 weeks, from about 1 week to about 3 months, from about 1 monthto about 6 months, from about 3 months to about 1 year, from about 9months to about 2 years, or more than 2 years.

In some embodiments, payload release may be consistent with nearzero-order kinetics. In some embodiments, payload release may beconsistent with first-order kinetics. In some embodiments, payloadrelease may be modulated based on the density, loading, molecularweight, and/or concentration of the payload. Where the carrier is anSBP, payload release may be modulated by one or more of SBP dryingmethod, silk fibroin molecular weight, and silk fibroin concentration.

In some embodiments, SBPs maintain and/or improve cargo stability,purity, and/or integrity. For example, SBPs may be used to protecttherapeutic agents or macromolecules during lyophilization. Themaintenance and/or improvement of stability during lyophilization may bedetermined by comparing SBP cargo stability to formulations lackingprocessed silk or to standard formulations in the art.

Viscosity

In some embodiments, SBPs may be formulated to modulate SBP viscosity.As used herein, the term “viscosity” refers to a measure of a material'sresistance to flow. The viscosity of a composition (e.g., a gel orhydrogel) provided herein can be determined using a rotationalviscometer or rheometer. Additional methods for determining theviscosity of a composition and other gel or rheological properties mayinclude any of those known in the art. In some embodiments, the SBPviscosity may be controlled via the concentration of processed silk. Insome embodiments, the SBP viscosity may be controlled via the molecularweight of processed silk. In some embodiments, the SBP viscosity may becontrolled via the boiling time of the processed silk. In someembodiments, SBP viscosity is altered by the incorporation of anexcipient. In some embodiments, SBP viscosity may be altered by theincorporation of an excipient that is a gelling agent. In someembodiments, the identity of the excipient (e.g., PEG or poloxamer) maybe altered to modulate SBP viscosity. In some embodiments, the viscosityof SBPs may be tuned for the desired application (e.g., drug deliverysystem, surgical implant, etc.). In some embodiments, the viscosity ofSBPs is tunable between 1-1000 centipoise (cP). In some embodiments, theviscosity of an SBP is tunable from about 0.0001 to about 1000 Pascalseconds (Pa*s). In some embodiments, the viscosity of an SBP is fromabout 1 cP to about 10 cP, from about 2 cP to about 20 cP, from about 3cP to about 30 cP, from about 4 cP to about 40 cP, from about 5 cP toabout 50 cP, from about 6 cP to about 60 cP, from about 7 cP to about 70cP, from about 8 cP to about 80 cP, from about 9 cP to about 90 cP, fromabout 10 cP to about 100 cP, from about 100 cP to about 150 cP, fromabout 150 cP to about 200 cP, from about 200 cP to about 250 cP, fromabout 250 cP to about 300 cP, from about 300 cP to about 350 cP, fromabout 350 cP to about 400 cP, from about 400 cP to about 450 cP, fromabout 450 cP to about 500 cP, from about 500 cP to about 600 cP, fromabout 550 cP to about 700 cP, from about 600 cP to about 800 cP, fromabout 650 cP to about 900 cP, or from about 700 cP to about 1000 cP. Insome embodiments, the viscosity of an SBP is from about 0.0001 Pa*s toabout 0.001 Pa*s, from about 0.001 Pa*s to about 0.01 Pa*s, from about0.01 Pa*s to about 0.1 Pa*s, from about 0.1 Pa*s to about 1 Pa*s, fromabout 1 Pa*s to about 10 Pa*s, from about 2 Pa*s to about 20 Pa*s, fromabout 3 Pa*s to about 30 Pa*s from about 4 Pa*s to about 40 Pa*s, fromabout 5 Pa*s to about 50 Pa*s, from about 6 Pa*s to about 60 Pa*s, fromabout 7 Pa*s to about 70 Pa*s, from about 8 Pa*s to about 80 Pa*s, fromabout 9 Pa*s to about 90 Pa*s, from about 10 Pa*s to about 100 Pa*s,from about 100 Pa*s to about 150 Pa*s, from about 150 Pa*s to about 200Pa*s, from about 200 Pa*s to about 250 Pa*s, from about 250 Pa*s toabout 300 Pa*s, from about 300 Pa*s to about 350 Pa*s, from about 350Pa*s to about 400 Pa*s, from about 400 Pa*s to about 450 Pa*s, fromabout 450 Pa*s to about 500 Pa*s, from about 500 Pa*s to about 600 Pa*s,from about 550 Pa*s to about 700 Pa*s, from about 600 Pa*s to about 800Pa*s, from about 650 Pa*s to about 900 Pa*s, from about 700 Pa*s toabout 1000 Pa*s or from about 10 Pa*s to about 2500 Pa*s. In someembodiments, the processed silk preparations may shear thin or displayshear thinning properties. As used herein, the term “shear thinning”refers to a decrease in viscosity at increasing shear rates. As usedherein, the term “shear rate” refers to the rate of change in the ratioof displacement of material upon the application of a shear force to theheight of the material. This ratio is also known as strain.

Stress Resistance

In some embodiments, SBPs may be formulated to modulate SBP resistanceto stress. Resistance to stress may be measured using one or morerheological measurements. Such measurements may include, but are notlimited to tensile elasticity, shear or rigidity, volumetric elasticity,and compression. Additional rheological measurements and properties mayinclude any of those taught in Zhang et a. (2017) Fiber and Polymers18(10):1831-1840; McGill et al. (2017) Acta Biomaterialia 63::76-84; andChoi et al. (2015) In-Situ Gelling Polymers, Series in BioEngineeringdoi. 10.1007/978-981-287-152-7_2, the contents of each of which areherein incorporated by reference in their entirety. In some embodiments,stress resistance may be modulated through incorporation of excipients(e.g., PEG or poloxamer). In some embodiments, SBP stress-resistanceproperties may be modulated to suit a specific application (e.g., tissueengineering scaffold, drug delivery system, surgical implant, etc.).

Concentrations and Ratios of SBP Components

SBPs may include formulations of processed silk with other components(e.g., excipients and cargo), wherein each SBP component is present at aspecific concentration, ratio, or range of concentrations or ratios,depending on SBP format and/or application. In some embodiments, theconcentration of processed silk or other SBP component (e.g., excipientor cargo) is present in SBPs at a concentration of from about 0.01%(w/v) to about 1% (w/v), from about 0.05% (w/v) to about 2% (w/v), fromabout 1% (w/v) to about 5% (w/v), from about 2% (w/v) to about 10%(w/v), from about 4% (w/v) to about 16% (w/v), from about 5% (w/v) toabout 20% (w/v), from about 8% (w/v) to about 24% (w/v), from about 10%(w/v) to about 30% (w/v), from about 12% (w/v) to about 32% (w/v), fromabout 14% (w/v) to about 34% (w/v), from about 16% (w/v) to about 36%(w/v), from about 18% (w/v) to about 38% (w/v), from about 20% (w/v) toabout 40% (w/v), from about 22% (w/v) to about 42% (w/v), from about 24%(w/v) to about 44% (w/v), from about 26% (w/v) to about 46% (w/v), fromabout 28% (w/v) to about 48% (w/v), from about 30% (w/v) to about 50%(w/v), from about 35% (w/v) to about 55% (w/v), from about 40% (w/v) toabout 60% (w/v), from about 45% (w/v) to about 65% (w/v), from about 50%(w/v) to about 70% (w/v), from about 55% (w/v) to about 75% (w/v), fromabout 60% (w/v) to about 80% (w/v), from about 65% (w/v) to about 85%(w/v), from about 70% (w/v) to about 90% (w/v), from about 75% (w/v) toabout 95% (w/v), from about 80% (w/v) to about 96% (w/v), from about 85%(w/v) to about 97% (w/v), from about 90%4 (w/v) to about 98% (w/v), fromabout 95% (w/v) to about 99% (w/v), from about 96% (w/v) to about 99.2%(w/v), from about 97% (w/v) to about 99.5% (w/v), from about 98% (w/v)to about 99.8% (w/v), from about 99% (w/v) to about 99.9% (w/v), orgreater than 99.9% (w/v).

In some embodiments, the concentration of processed silk or other SBPcomponent (e.g., excipient or cargo) is present in SBPs at aconcentration of from about 0.01% (v/v) to about 1% (v/v), from about0.05% (v/v) to about 2% (v/v), from about 1% (v/v) to about 5% (v/v),from about 2% (v/v) to about 10% (v/v), from about 4% (v/v) to about 16%(v/v), from about 5% (v/v) to about 20% (v/v), from about 8% (v/v) toabout 24% (v/v), from about 10% (v/v) to about 30% (v/v), from about 12%(v/v) to about 32% (v/v), from about 14% (v/v) to about 34% (v/v), fromabout 16% (v/v) to about 36% (v/v), from about 18% (v/v) to about 38%(v/v), from about 20% (v/v) to about 40% (v/v), from about 22% (v/v) toabout 42% (v/v), from about 24% (v/v) to about 44% (v/v), from about 26%(v/v) to about 46% (v/v), from about 28% (v/v) to about 48% (v/v), fromabout 30% (v/v) to about 50% (v/v), from about 35% (v/v) to about 55%(v/v), from about 40% (v/v) to about 60% (v/v), from about 45% (v/v) toabout 65% (v/v), from about 50% (v/v) to about 70% (v/v), from about 55%(v/v) to about 75% (v/v), from about 60% (v/v) to about 80% (v/v), fromabout 65% (v/v) to about 85% (v/v), from about 70% (v/v) to about 90%(v/v), from about 75% (v/v) to about 95% (v/v), from about 80% (v/v) toabout 96% (v/v), from about 85% (v/v) to about 97% (v/v), from about 90%(v/v) to about 98% (v/v), from about 95% (v/v) to about 99% (v/v), fromabout 96% (v/v) to about 99.2% (v/v), from about 97% (v/v) to about99.5% (v/v), from about 98% (v/v) to about 99.8% (v/v), from about 99%(v/v) to about 99.9% (v/v), or greater than 99.9% (v/v).

In one embodiment, the concentration of processed silk or other SBPcomponent (e.g., excipient or cargo) is present in SBPs at aconcentration of 1% (w/v).

In one embodiment, the concentration of processed silk or other SBPcomponent (e.g., excipient or cargo) is present in SBPs at aconcentration of 2% (w/v).

In one embodiment, the concentration of processed silk or other SBPcomponent (e.g., excipient or cargo) is present in SBPs at aconcentration of 3% (w/v).

In one embodiment, the concentration of processed silk or other SBPcomponent (e.g., excipient or cargo) is present in SBPs at aconcentration of 4% (w/v).

In one embodiment, the concentration of processed silk or other SBPcomponent (e.g., excipient or cargo) is present in SBPs at aconcentration of 5% (w/v).

In one embodiment, the concentration of processed silk or other SBPcomponent (e.g., excipient or cargo) is present in SBPs at aconcentration of 6% (w/v).

In one embodiment, the concentration of processed silk or other SBPcomponent (e.g., excipient or cargo) is present in SBPs at aconcentration of 10% (w/v).

In one embodiment, the concentration of processed silk or other SBPcomponent (e.g., excipient or cargo) is present in SBPs at aconcentration of 20% (w/v).

In one embodiment, the concentration of processed silk or other SBPcomponent (e.g., excipient or cargo) is present in SBPs at aconcentration of 30% (w/v).

In one embodiment, the concentration of processed silk or other SBPcomponent (e.g., excipient or cargo) is present in SBPs at aconcentration of 16.7% (w/w).

In one embodiment, the concentration of processed silk or other SBPcomponent (e.g., excipient or cargo) is present in SBPs at aconcentration of 20% (w/w).

In one embodiment, the concentration of processed silk or other SBPcomponent (e.g., excipient or cargo) is present in SBPs at aconcentration of 23% (w/w).

In one embodiment, the concentration of processed silk or other SBPcomponent (e.g., excipient or cargo) is present in SBPs at aconcentration of 25% (w/w).

In one embodiment, the concentration of processed silk or other SBPcomponent (e.g., excipient or cargo) is present in SBPs at aconcentration of 27.3% (w/w).

In one embodiment, the concentration of processed silk or other SBPcomponent (e.g., excipient or cargo) is present in SBPs at aconcentration of 28.6% (w/w).

In one embodiment, the concentration of processed silk or other SBPcomponent (e.g., excipient or cargo) is present in SBPs at aconcentration of 33.3% (w/w).

In one embodiment, the concentration of processed silk or other SBPcomponent (e.g., excipient or cargo) is present in SBPs at aconcentration of 40% (w/v).

In one embodiment, the concentration of processed silk or other SBPcomponent (e.g., excipient or cargo) is present in SBPs at aconcentration of 50% (w/w).

In some embodiments, the concentration of processed silk or other SBPcomponent (e.g., excipient or cargo) is present in SBPs at aconcentration of from about 0.01% (w/w) to about 1% (w/w), from about0.05% (w/w) to about 2% (w/w), from about 1% (w/w) to about 5% (w/w),from about 2% (w/w) to about 10% (w/w), from about 4% (w/w) to about 16%(w/w), from about 5% (w/w) to about 20% (w/v), from about 8% (w/w) toabout 24% (w/w), from about 10% (w/w) to about 30% (w/w), from about 12%(w/w) to about 32% (w/w), from about 14% (w/w) to about 34% (w/w), fromabout 16% (w/v) to about 36% (w/v), from about 18% (w/w) to about 38%(w/w), from about 20% (w/v) to about 40% (w/v), from about 22% (w/w) toabout 42% (w/w), from about 24% (w/v) to about 44% (w/w), from about 26%(w/v) to about 46% (w/w), from about 28% (w/w) to about 48% (w/w), fromabout 30% (w/w) to about 50% (w/w), from about 35% (w/w) to about 55%(w/w), from about 40% (w/w) to about 60% (w/w), from about 45% (w/w) toabout 65% (w/w), from about 50% (w/w) to about 70% (w/w), from about 55%(w/w) to about 75% (w/w), from about 60% (w/w) to about 80% (w/w), fromabout 65% (w/w) to about 85% (w/w), from about 70% (w/w) to about 90%(w/w), from about 75% (w/w) to about 95% (w/w), from about 80% (w/v) toabout 96% (w/v), from about 85% (w/w) to about 97% (w/w), from about 90%(w/v) to about 98% (w/w), from about 95% (w/v) to about 99% (w/w), fromabout 96% (w/w) to about 99.2% (w/w), from about 97% (w/w) to about99.5% (w/w), from about 98% (w/w) to about 99.8% (w/w), from about 99%(w/w) to about 99.9% (w/w), or greater than 99.9% (w/w).

In some embodiments, the concentration of processed silk (e.g., silkfibroin) or other SBP component (e.g., excipient or cargo) is present inSBPs at a concentration of from about 0.01 pg/mL to about 1 pg/mL, fromabout 0.05 pg/mL to about 2 pg/mL, from about 1 pg/mL to about 5 pg/mL,from about 2 pg/mL to about 10 pg/mL, from about 4 pg/mL to about 16pg/mL, from about 5 pg/mL to about 20 pg/mL, from about 8 pg/mL to about24 pg/mL, from about 10 pg/mL to about 30 pg/mL, from about 12 pg/mL toabout 32 pg/mL, from about 14 pg/mL to about 34 pg/mL, from about 16pg/mL to about 36 pg/mL, from about 18 pg/mL to about 38 pg/mL, fromabout 20 pg/mL to about 40 pg/mL, from about 22 pg/mL to about 42 pg/mL,from about 24 pg/mL to about 44 pg/mL, from about 26 pg/mL to about 46pg/mL, from about 28 pg/mL to about 48 pg/mL, from about 30 pg/mL toabout 50 pg/mL, from about 35 pg/mL to about 55 pg/mL, from about 40pg/mL to about 60 pg/mL, from about 45 pg/mL to about 65 pg/mL, fromabout 50 pg/mL to about 75 pg/mL, from about 60 pg/mL to about 240pg/mL, from about 70 pg/mL to about 350 pg/mL, from about 80 pg/mL toabout 400 pg/mL, from about 90 pg/mL to about 450 pg/mL, from about 100pg/mL to about 500 pg/mL, from about 0.01 ng/mL to about 1 ng/mL, fromabout 0.05 ng/mL to about 2 ng/mL, from about 1 ng/mL to about 5 ng/mL,from about 2 ng/mL to about 10 ng/mL, from about 4 ng/mL to about 16ng/mL, from about 5 ng/mL to about 20 ng/mL, from about 8 ng/mL to about24 ng/mL, from about 10 ng/mL to about 30 ng/mL, from about 12 ng/mL toabout 32 ng/mL, from about 14 ng/mL to about 34 ng/mL, from about 16ng/mL to about 36 ng/mL, from about 18 ng/mL to about 38 ng/mL, fromabout 20 ng/mL to about 40 ng/mL, from about 22 ng/mL to about 42 ng/mL,from about 24 ng/mL to about 44 ng/mL, from about 26 ng/mL to about 46ng/mL, from about 28 ng/mL to about 48 ng/mL, from about 30 ng/mL toabout 50 ng/mL, from about 35 ng/mL to about 55 ng/mL, from about 40ng/mL to about 60 ng/mL, from about 45 ng/mL to about 65 ng/mL, fromabout 50 ng/mL to about 75 ng/mL, from about 60 ng/mL to about 240ng/mL, from about 70 ng/mL to about 350 ng/mL, from about 80 ng/mL toabout 400 ng/mL, from about 90 ng/mL to about 450 ng/mL, from about 100ng/mL to about 500 ng/mL, from about 0.01 μg/mL to about 1 μg/mL, fromabout 0.05 μg/mL to about 2 μg/mL, from about 1 μg/mL to about 5 μg/mL,from about 2 μg/mL to about 10 μg/mL, from about 4 μg/mL to about 16μg/mL, from about 5 μg/mL to about 20 μg/mL, from about 8 μg/mL to about24 μg/mL, from about 10 μg/mL to about 30 μg/mL, from about 12 μg/mL toabout 32 μg/mL, from about 14 μg/mL to about 34 μg/mL, from about 16μg/mL to about 36 μg/mL, from about 18 μg/mL to about 38 μg/mL, fromabout 20 μg/mL to about 40 μg/mL, from about 22 μg/mL to about 42 μg/mL,from about 24 μg/mL to about 44 μg/mL, from about 26 μg/mL to about 46μg/mL, from about 28 μg/mL to about 48 μg/mL, from about 30 μg/mL toabout 50 μg/mL, from about 35 μg/mL to about 55 μg/mL, from about 40μg/mL to about 60 μg/mL, from about 45 μg/mL to about 65 μg/mL, fromabout 50 μg/mL to about 75 μg/mL, from about 60 μg/mL to about 240μg/mL, from about 70 μg/mL to about 350 μg/mL, from about 80 μg/mL toabout 400 μg/mL, from about 90 μg/mL to about 450 μg/mL, from about 100μg/mL to about 500 μg/mL, from about 0.01 mg/mL to about 1 mg/mL, fromabout 0.05 mg/mL to about 2 mg/mL, from about 1 mg/mL to about 5 mg/mL,from about 2 mg/mL to about 10 mg/mL, from about 4 mg/mL to about 16mg/mL, from about 5 mg/mL to about 20 mg/mL, from about 8 mg/mL to about24 mg/mL, from about 10 mg/mL to about 30 mg/mL, from about 12 mg/mL toabout 32 mg/mL, from about 14 mg/mL to about 34 mg/mL, from about 16mg/mL to about 36 mg/mL, from about 18 mg/mL to about 38 mg/mL, fromabout 20 mg/mL to about 40 mg/mL, from about 22 mg/mL to about 42 mg/mL,from about 24 mg/mL to about 44 mg/mL, from about 26 mg/mL to about 46mg/mL, from about 28 mg/mL to about 48 mg/mL, from about 30 mg/mL toabout 50 mg/mL, from about 35 mg/mL to about 55 mg/mL, from about 40mg/mL to about 60 mg/mL, from about 45 mg/mL to about 65 mg/mL, fromabout 50 mg/mL to about 75 mg/mL, from about 60 mg/mL to about 240mg/mL, from about 70 mg/mL to about 350 mg/mL, from about 80 mg/mL toabout 400 mg/mL, from about 90 mg/mL to about 450 mg/mL, from about 100mg/mL to about 500 mg/mL, from about 0.01 g/mL to about 1 g/mL, fromabout 0.05 g/mL to about 2 g/mL, from about 1 g/mL to about 5 g/mL, fromabout 2 g/mL to about 10 g/mL, from about 4 g/mL to about 16 g/mL, orfrom about 5 g/mL to about 20 g/mL.

In one embodiment, the concentration of processed silk (e.g., silkfibroin) or other SBP component (e.g., excipient or cargo) is present inSBPs at a concentration of 5 mg/mL.

In one embodiment, the concentration of processed silk (e.g., silkfibroin) or other SBP component (e.g., excipient or cargo) is present inSBPs at a concentration of 2.5 mg/mL.

In one embodiment, the concentration of processed silk (e.g., silkfibroin) or other SBP component (e.g., excipient or cargo) is present inSBPs at a concentration of 1.25 mg/mL.

In one embodiment, the concentration of processed silk (e.g., silkfibroin) or other SBP component (e.g., excipient or cargo) is present inSBPs at a concentration of 0.625 mg/mL.

In one embodiment, the concentration of processed silk (e.g., silkfibroin) or other SBP component (e.g., excipient or cargo) is present inSBPs at a concentration of 0.3125 mg/mL.

In some embodiments, the concentration of processed silk (e.g., silkfibroin) or other SBP component (e.g., excipient or cargo) is present inSBPs at a concentration of from about 0.01 pg/kg to about 1 pg/kg, fromabout 0.05 pg/kg to about 2 pg/kg, from about 1 pg/kg to about 5 pg/kg,from about 2 pg/kg to about 10 pg/kg, from about 4 pg/kg to about 16pg/kg, from about 5 pg/kg to about 20 pg/kg, from about 8 pg/kg to about24 pg/kg, from about 10 pg/kg to about 30 pg/kg, from about 12 pg/kg toabout 32 pg/kg, from about 14 pg/kg to about 34 pg/kg, from about 16pg/kg to about 36 pg/kg, from about 18 pg/kg to about 38 pg/kg, fromabout 20 pg/kg to about 40 pg/kg, from about 22 pg/kg to about 42 pg/kg,from about 24 pg/kg to about 44 pg/kg, from about 26 pg/kg to about 46pg/kg, from about 28 pg/kg to about 48 pg/kg, from about 30 pg/kg toabout 50 pg/kg, from about 35 pg/kg to about 55 pg/kg, from about 40pg/kg to about 60 pg/kg, from about 45 pg/kg to about 65 pg/kg, fromabout 50 pg/kg to about 75 pg/kg, from about 60 pg/kg to about 240pg/kg, from about 70 pg/kg to about 350 pg/kg, from about 80 pg/kg toabout 400 pg/kg, from about 90 pg/kg to about 450 pg/kg, from about 100pg/kg to about 500 pg/kg, from about 0.01 ng/kg to about 1 ng/kg, fromabout 0.05 ng/kg to about 2 ng/kg, from about 1 ng/kg to about 5 ng/kg,from about 2 ng/kg to about 10 ng/kg, from about 4 ng/kg to about 16ng/kg, from about 5 ng/kg to about 20 ng/kg, from about 8 ng/kg to about24 ng/kg, from about 10 ng/kg to about 30 ng/kg, from about 12 ng/kg toabout 32 ng/kg, from about 14 ng/kg to about 34 ng/kg, from about 16ng/kg to about 36 ng/kg, from about 18 ng/kg to about 38 ng/kg, fromabout 20 ng/kg to about 40 ng/kg, from about 22 ng/kg to about 42 ng/kg,from about 24 ng/kg to about 44 ng/kg, from about 26 ng/kg to about 46ng/kg, from about 28 ng/kg to about 48 ng/kg, from about 30 ng/kg toabout 50 ng/kg, from about 35 ng/kg to about 55 ng/kg, from about 40ng/kg to about 60 ng/kg, from about 45 ng/kg to about 65 ng/kg, fromabout 50 ng/kg to about 75 ng/kg, from about 60 ng/kg to about 240ng/kg, from about 70 ng/kg to about 350 ng/kg, from about 80 ng/kg toabout 400 ng/kg, from about 90 ng/kg to about 450 ng/kg, from about 100ng/kg to about 500 ng/kg, from about 0.01 μg/kg to about 1 μg/kg, fromabout 0.05 μg/kg to about 2 μg/kg, from about 1 μg/kg to about 5 μg/kg,from about 2 μg/kg to about 10 μg/kg, from about 4 μg/kg to about 16μg/kg, from about 5 μg/kg to about 20 μg/kg, from about 8 μg/kg to about24 μg/kg, from about 10 μg/kg to about 30 μg/kg, from about 12 μg/kg toabout 32 μg/kg, from about 14 μg/kg to about 34 μg/kg, from about 16μg/kg to about 36 μg/kg, from about 18 μg/kg to about 38 μg/kg, fromabout 20 μg/kg to about 40 μg/kg, from about 22 μg/kg to about 42 μg/kg,from about 24 μg/kg to about 44 μg/kg, from about 26 μg/kg to about 46μg/kg, from about 28 μg/kg to about 48 μg/kg, from about 30 μg/kg toabout 50 μg/kg, from about 35 μg/kg to about 55 μg/kg, from about 40μg/kg to about 60 μg/kg, from about 45 μg/kg to about 65 μg/kg, fromabout 50 μg/kg to about 75 μg/kg, from about 60 μg/kg to about 240μg/kg, from about 70 μg/kg to about 350 μg/kg, from about 80 μg/kg toabout 400 μg/kg, from about 90 μg/kg to about 450 μg/kg, from about 100μg/kg to about 500 μg/kg, from about 0.01 mg/kg to about 1 mg/kg, fromabout 0.05 mg/kg to about 2 mg/kg, from about 1 mg/kg to about 5 mg/kg,from about 2 mg/kg to about 10 mg/kg, from about 4 mg/kg to about 16mg/kg, from about 5 mg/kg to about 20 mg/kg, from about 8 mg/kg to about24 mg/kg, from about 10 mg/kg to about 30 mg/kg, from about 12 mg/kg toabout 32 mg/kg, from about 14 mg/kg to about 34 mg/kg, from about 16mg/kg to about 36 mg/kg, from about 18 mg/kg to about 38 mg/kg, fromabout 20 mg/kg to about 40 mg/kg, from about 22 mg/kg to about 42 mg/kg,from about 24 mg/kg to about 44 mg/kg, from about 26 mg/kg to about 46mg/kg, from about 28 mg/kg to about 48 mg/kg, from about 30 mg/kg toabout 50 mg/kg, from about 35 mg/kg to about 55 mg/kg, from about 40mg/kg to about 60 mg/kg, from about 45 mg/kg to about 65 mg/kg, fromabout 50 mg/kg to about 75 mg/kg, from about 60 mg/kg to about 240mg/kg, from about 70 mg/kg to about 350 mg/kg, from about 80 mg/kg toabout 400 mg/kg, from about 90 mg/kg to about 450 mg/kg, from about 100mg/kg to about 500 mg/kg, from about 0.01 g/kg to about 1 g/kg, fromabout 0.05 g/kg to about 2 g/kg, from about 1 g/kg to about 5 g/kg, fromabout 2 g/kg to about 10 g/kg, from about 4 g/kg to about 16 g/kg, orfrom about 5 g/kg to about 20 g/kg, from about 10 g/kg to about 50 g/kg,from about 15 g/kg to about 100 g/kg, from about 20 g/kg to about 150g/kg, from about 25 g/kg to about 200 g/kg, from about 30 g/kg to about250 g/kg, from about 35 g/kg to about 300 g/kg, from about 40 g/kg toabout 350 g/kg, from about 45 g/kg to about 400 g/kg, from about 50 g/kgto about 450 g/kg, from about 55 g/kg to about 500 g/kg, from about 60g/kg to about 550 g/kg, from about 65 g/kg to about 600 g/kg, from about70 g/kg to about 650 g/kg, from about 75 g/kg to about 700 g/kg, fromabout 80 g/kg to about 750 g/kg, from about 85 g/kg to about 800 g/kg,from about 90 g/kg to about 850 g/kg, from about 95 g/kg to about 900g/kg, from about 100 g/kg to about 950 g/kg, or from about 200 g/kg toabout 1000 g/kg.

In some embodiments, SBPs may be formatted as a gel. Such gels mayinclude hydrogels. In some embodiments, such hydrogels are formulatedwith therapeutic agents. Therapeutic agents may include a nonsteroidalanti-inflammatory drug (NSAID), for example, celecoxib.

Appearance: Transparent, Opaque, Translucent

In some embodiments, the appearance of SBPs described in the presentdisclosure may be tuned for the application for which they weredesigned. In some embodiments, SBPs may be transparent. In someembodiments, SBPs may be translucent. In some embodiments, SBPs may beopaque. In some embodiments, SBP preparation methods may be used tomodulate clarity, as taught in International Patent ApplicationPublication No. WO2012170655, the contents of which are hereinincorporated by reference in their entirety. In some embodiments, theincorporation of excipients may be used to tune the clarity of processedsilk preparations. In some embodiments, the excipient is sucrose. Insome embodiments, the sucrose may also increase protein reconstitutionduring lyophilization. In some embodiments, sucrose may improveprocessed silk hydrogel clarity (optical transparency). In someembodiments, optically transparent SBPs may be used for ocularapplications, e.g., treatment of ocular conditions, diseases, and/orindications. In some embodiments, SBPs herein may be used to labelproducts, as taught in International Patent Application Publication No.WO2009155397, the contents of which are herein incorporated by referencein their entirety. The transparency of SBPs, as well as otherproperties, may render resulting labels edible, biodegradable, and/orholographic.

Residence Time

In some embodiments, SBP formulations may be prepared to have desiredresidence time according to the application for which they are designed.As used herein, the term “residence time” refers to the average lengthof time during which a substance (e.g., SBP formulations) is in a givenlocation or condition. In some embodiments, residence time of SBPformulations described herein may vary from a few hours to severalmonths. For example, residence time of SBP formulations may be about 1hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours,about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours,about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 1day, about 2 days, about 3 days, about 4 days, about 5 days, about 6days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1month, about 2 months, about 3 months, about 4 months, about 5 months,about 6 months, about 7 months, about 8 months, about 9 months, about 10months, about 11 months, or longer than 1 year.

pH

SBPs may have a pH from about 3 to about 10. In some embodiments, the pHis from about 3 to about 6, from about 6 to about 8, or from about 8 toabout 10. In some embodiments, the pH of the SBP is about 7.4. In someembodiments, the pH of the SBP is 7.06. In some embodiments, the pH ofthe SBP is 7.15.

Exemplary Formulations

In one embodiment, the SBP formulation may include 480 mb silk fibroinat a concentration of 3%, an excipient at a concentration of 10% andcargo at a concentration of 10%. The excipient cargo may be, but is notlimited to, poloxamer-188 (P188) and PEG4k, and the cargo may be,celecoxib (CXB), bovine serum albumin (BSA), lysozyme or bevacizumab.The osmolarity of the SBP formulation may be the range of 290-320mOsm/L.

In one embodiment, the SBP formulation may include 480 mb silk fibroinat a concentration of 3%, an excipient at a concentration of 20% andcargo at a concentration of 1%. The excipient cargo may be, but is notlimited to, poloxamer-188 (P188) and PEG4k, and the cargo may be,celecoxib (CXB), bovine serum albumin (BSA), lysozyme or bevacizumab.The osmolarity of the SBP formulation may be the range of 290-320mOsm/L.

In one embodiment, the SBP formulation may include 480 mb silk fibroinat a concentration of 3%, an excipient at a concentration of 50% andcargo at a concentration of 1%. The excipient cargo may be, but is notlimited to, poloxamer-188 (P188) and PEG4k, and the cargo may be,celecoxib (CXB), bovine serum albumin (BSA), lysozyme or bevacizumab.The osmolarity of the SBP formulation may be the range of 290-320mOsm/L.

In one embodiment, the SBP formulation may include 120 mb silk fibroinat a concentration of 2%, 3%, 4%, 5%, or 6%. The SBP formulation mayinclude an excipient at a concentration of 40% and may be PEG300 orglycerol and/or cargo a concentration of 10%. The cargo may be,celecoxib (CXB), bovine serum albumin (BSA), lysozyme or bevacizumab.Additionally 0.2% polysorbate-80 and 22 mM phosphate buffer may beincluded in the formulation.

Combinations

In some embodiments, SBPs are presented in a combinatorial format. Acombinatorial format may consist of two or more different materials thathave been combined to form a single composition. In some embodiments,two or more SBPs of different formats (e.g. rod, hydrogel etc.) arecombined to form a single composition (e.g., see European PublicationNumber EP3212246, the contents of which are herein incorporated byreference in their entirety). In some embodiments, one or more SBP iscombined with a different material (e.g. a polymer, a mat, a particle, amicrosphere, a nanosphere, a metal, a scaffold, etc.) to form a singlecomposition (e.g., see International Publication Number WO2017179069,the contents of which are herein incorporated by reference in theirentirety. In some embodiments, combinatorial formats are prepared byformulating two or more SBPs of different formats as a singlecomposition (e.g., see Kambe et al. (2017) Materials (Basel)10(10):1153, the contents of which are herein incorporated by referencein their entirety). In some embodiments, combinatorial formats areprepared by formulating two or more SBPs of different formats, alongwith another material, as a single composition (e.g., see InternationalPublication Number WO2017177281, the contents of which are hereinincorporated by reference in their entirety). In some embodiments,combinatorial formats include adding one or more SBPs to a first SBP ofa different format (e.g., see European Patent Number EP3212246, thecontents of which are herein incorporated by reference in theirentirety). In some embodiments, combinatorial formats include adding oneor more SBPs to a first composition comprising a different material(e.g., see Jiang et al. (2017) J Biomater Sci Polym Ed 15:1-36, thecontents of which are herein incorporated by reference in theirentirety). In some embodiments, the combinatorial formats are preparedby adding one or more materials to one or more first formed SBPs (e.g.,see Babu et al. (2017) J Colloid Interface Sci 513:62-72, the contentsof which are herein incorporated by reference in their entirety).

Distribution

SBP components may be distributed equally or unequally, depending onformat and application. Non-limiting examples of unequal distributioninclude component localization in SBP regions or compartments, on SBPsurfaces, etc. In some embodiments, components include cargo. Such cargomay include payloads, for example, therapeutic agents. In someembodiments, therapeutic agents are present on the surface of an SBP(e.g., see Han et al. (2017) Biomacromolecules 18(11):3776-3787.; Ran etal. (2017) Biomacromolecules 18(11):3788-3801, the contents of each ofwhich are herein incorporated by reference in their entirety). In someembodiments, components (e.g., therapeutic agents) are homogenouslymixed with processed silk to generate a desired distribution (e.g., seeUnited States Publication No. US20170333351; Sun et al. (2017) Journalof Materials Chemistry B 5:8770-8779; and Du et al. (2017) Nanoscale ResLett 12(1):573, the contents of each of which are herein incorporated byreference in their entirety). In some embodiments, components (e.g.,therapeutic agents) are encapsulated in SBPs (e.g., see Shi et al.(2017) Nanoscale 9:14520, the contents of which are herein incorporatedby reference in their entirety).

Solubility

In some embodiments, SBPs or components thereof are water soluble. Thewater solubility, along with the rate of degradation, of SBPs maymodulate payload (e.g., therapeutic agent) release rate and/or releaseperiod. An increasing amount of payload may be released into surroundingmedium as surrounding matrix dissolves (e.g., see InternationalPublication Numbers WO2013126799 and WO2017165922; and U.S. Pat. No.8,530,625, the contents of each of which are herein incorporated byreference in their entirety). Longer time periods required to dissolveSBPs or components thereof may result in longer release periods. In someembodiments, SBP solubility may be modulated in order to control therate of payload release in the surrounding medium. The solubility ofSBPs may be modulated via any method known to those skilled in the art.In some embodiments, SBP solubility may be modulated by alteringincluded silk fibroin secondary structure (e.g., increasing 0-sheetcontent and/or crystallinity). In some embodiments, SBP solubility maybe modulated by altering SBP format. In some embodiments, SBP solubilityand/or rate of degradation may be modulated to facilitate extendedrelease of therapeutic agent payloads in vitro and/or in vivo.

Coating Agents

In some embodiments, SBPs may be used as coating agents. As used herein,the term “coating agent” refers to a substance covering or used to coveran article, wherein the substance adheres to the article (also referredto herein as “coatings”). SBP coating agents may be used, for example,to coat cargo, payloads, devices, or device components. SBP coatings mayinclude therapeutic agent payloads (e.g., ocular therapeutic agents) andmay be used to coat articles (e.g., implants) used to deliver suchtherapeutic agent payloads.

Rods

In some embodiments, SBPs are prepared as rods. As used herein whenreferring to processed silk preparations or SBPs, the term “rod” refersto an elongated format, typically cylindrical, that may have blunted ortapered ends. Rods may be suitable for implantation or similaradministration methods as it may be possible to deliver rods byinjection. Rods may also be obtained simply by passing suitably viscousprocessed silk preparations through a needle, cannula, tube, or opening.In some embodiments, rods are prepared by one or more of injectionmolding, heated or cooled extrusion, extrusion through a coating agent,milling with a therapeutic agent, and combining with a polymer followedby extrusion.

In some embodiments, SBP rods include processed silk (e.g., silkfibroin) rods. Some rods may include coterminous luminal cavities inwhole or in part running through the rod. Rods may be of anycross-sectional shape, including, but not limited to, circular, square,oval, triangular, irregular, or combinations thereof.

In some embodiments, rods are prepared from silk fibroin preparations.The silk fibroin preparations may include lyophilized silk fibroin. Thelyophilized silk fibroin may be dissolved in water to form silk fibroinsolutions used in rod preparation. Silk fibroin solutions may beprepared as stock solutions to be combined with additional componentsprior to rod preparation. In some embodiments silk fibroin stocksolutions have a silk fibroin concentration of between 10% (w/v) and 40%(w/v). In some embodiments, the silk fibroin stock solution for thepreparation of silk fibroin rods has a concentration of at least 10%(w/v), at least 20% (w/v), at least 30% (w/v), at least 40% (w/v), or atleast 50% (w/v).

In one embodiment, the silk fibroin stock solution has a concentrationof 10% (w/v).

In one embodiment, the silk fibroin stock solution has a concentrationof 20% (w/v).

In one embodiment, the silk fibroin stock solution has a concentrationof 30% (w/v).

In one embodiment, the silk fibroin stock solution has a concentrationof 40% (w/v).

In one embodiment, the silk fibroin stock solution has a concentrationof 50% (w/v).

In some embodiments, silk fibroin stock solution prepared for rodformation are mixed with one or more other components intended to beinclude in the final processed silk rods. Examples of such othercomponents include, but are not limited to, excipients, salts,therapeutic agents, biological agents, proteins, small molecules, andpolymers. In some embodiments, processed silk rods may include between20 to 55% (w/w) silk fibroin. In some embodiments, processed silk rodsmay include between 40 to 80% (w/w) therapeutic agent. In someembodiments, processed silk rods may include 35% (w/w) silk fibroin and65% (w/w) therapeutic agent. In some embodiments, processed silk rodsmay include 30% (w/w) silk fibroin and 70% (w/w) therapeutic agent. Insome embodiments, processed silk rods may include 40% (w/w) silk fibroinand 60% (w/w) therapeutic agent. In some embodiments, processed silkrods may include 26% (w/w) silk fibroin and 74% (w/w) therapeutic agent.In some embodiments, processed silk rods may include 37% (w/w) silkfibroin and 63% (w/w) therapeutic agent. In some embodiments, processedsilk rods may include 33% (w/w) silk fibroin and 66% (w/w) therapeuticagent. In some embodiments, processed silk rods may include 51% (w/w)silk fibroin and 49% (w/w) therapeutic agent. In some embodiments, thesilk fibroin may be included at a concentration (w/w) of 0.01% to about1%, from about 0.05% to about 2%, from about 0.1% to about 30%, fromabout 1% to about 5%, from about 2% to about 10%, from about 3% to about15%, from about 4% to about 20%, from about 5% to about 25%, from about6% to about 30%, from about 7% to about 35%, from about 8% to about 40%,from about 9% to about 45%, from about 10% to about 50%, from about 12%to about 55%, from about 14% to about 60%, from about 16% to about 65%,from about 18% to about 70%, from about 20% to about 75%, from about 22%to about 80%, from about 24% to about 85%, from about 26% to about 90%,from about 28% to about 95%, from about 30% to about 96%, from about 32%to about 97%, from about 34% to about 98%, from about 36% to about98.5%, from about 38% to about 99%, from about 40% to about 99.5%, fromabout 42% to about 99.6%, from about 44% to about 99.7%, from about 46%to about 99.8%, or from about 50% to about 99.9%.

In some embodiments, processed silk rods are prepared by extrusion. Asused herein, the term “extrusion” refers to a process by which asubstance is forced through an opening, tube, or passage. In someembodiments, processed silk rods are formed by extruding processed silkpreparations through a needle or cannula. Processed silk preparationsused for rod formation may have varying levels of viscosity. Preparationviscosity may depend on the presence and/or identity of excipientspresent. In some embodiments, processed silk preparations may includecompounds or compositions intended to be embedded in rods prepared byextrusion. Excipients, compounds, or compositions included in processedsilk preparations used for extrusion may include, but are not limitedto, salts, therapeutic agents, biological agents, proteins, smallmolecules, and polymers. Extrusion may be carried out manually or by anautomated process.

In some embodiments, extrusion may be carried out using a syringe. Thesyringe may be fitted with a needle, tube, or cannula. The needle, tube,or cannula may have a sharpened end or a blunt end. The needle may havea diameter of from about 0.1 mm to about 0.3 mm, from about 0.2 mm toabout 0.7 mm, from about 0.4 mm to about 1.1 mm, from about 0.6 mm toabout 1.5 mm, from about 0.8 mm to about 1.9 mm, from about 1 mm toabout 2.3 mm, from about 1.2 mm to about 2.7 mm, from about 1.6 mm toabout 3.1 mm, or from about 2 mm to about 3.5 mm. Processed silkpreparations may be used to fill tubes, wherein the processed silkpreparations are incubated in the tubes for various periods of timeunder various conditions (e.g., various temperatures). In someembodiments, tubing filled with processed silk preparation may beincubated at 37° C. for from about 2 hours to about 36 hours or more. Insome embodiments, processed silk filled tubing is incubated for 24hours. In some embodiments, processed silk preparations remain in tubingafter the 37° C. incubation. In some embodiments, processed silkpreparations are removed from the tubing after the incubation at 37° C.Processed silk preparations removed from tubing may maintain arod-shaped format. Such preparations may be dried after removal fromtubing. In some embodiments, processed silk preparations may be encasedin tubing while drying. Rods may be dried by one or more offreeze-drying, oven drying, and air drying. Some processed silkpreparations may be removed tubing after drying.

Tubing used for extrusion may be composed of various materials. In someembodiments, tubing is made from one or more of silicone,polyetheretherketone (PEEK), polytetrafluoroethylene (PTFE), amorphousfluoroplastics, fluorinated ethylene propylene, perfluoroalkoxycopolymers, ethylene-tetrafluoroethylene, polyolefins, and nylon.

In some embodiments, rods may have a diameter of from about 0.05 μm toabout 10 μm, from about 1 μm to about 20 μm, from about 2 μm to about 30μm, from about 5 μm to about 40 μm, from about 10 μm to about 50 μm,from about 20 μm to about 60 μm, from about 30 μm to about 70 μm, fromabout 40 μm to about 80 μm, from about 50 μm to about 90 μm, from about0.05 mm to about 2 mm, from about 0.1 mm to about 3 mm, from about 0.2mm to about 4 mm, from about 0.5 mm to about 5 mm, from about 1 mm toabout 6 mm, from about 2 mm to about 7 mm, from about 5 mm to about 10mm, from about 8 mm to about 16 mm, from about 10 mm to about 50 mm,from about 20 mm to about 100 mm, from about 40 mm to about 200 mm, fromabout 60 mm to about 300 mm, from about 80 mm to about 400 mm, fromabout 250 mm to about 750 mm, or from about 500 mm to about 1000 mm. Insome embodiments, rods include a diameter of at least 0.5 μm, at least 1μm, at least 10 μm, at least 100 μm, at least 500 μm, at least 1 mm, atleast 10 mm, or at least 100 mm. In one embodiment, the rods have adiameter of 1 mm. In another embodiment, the rods have a diameter of 0.5mm. In another embodiment, the rods have a diameter of 400 um. Inanother embodiment, the rods have a diameter of 430 um.

In some embodiments, the rods described herein may have a density offrom about 0.01 μg/mL to about 1 μg/mL, from about 0.05 μg/mL to about 2μg/mL, from about 1 μg/mL to about 5 μg/mL, from about 2 μg/mL to about10 μg/mL, from about 4 μg/mL to about 16 μg/mL, from about 5 μg/mL toabout 20 μg/mL, from about 8 μg/mL to about 24 μg/mL, from about 10μg/mL to about 30 μg/mL, from about 12 μg/mL to about 32 μg/mL, fromabout 14 μg/mL to about 34 μg/mL, from about 16 μg/mL to about 36 μg/mL,from about 18 μg/mL to about 38 μg/mL, from about 20 μg/mL to about 40μg/mL, from about 22 μg/mL to about 42 μg/mL, from about 24 μg/mL toabout 44 μg/mL, from about 26 μg/mL to about 46 μg/mL, from about 28μg/mL to about 48 μg/mL, from about 30 μg/mL to about 50 μg/mL, fromabout 35 μg/mL to about 55 μg/mL, from about 40 μg/mL to about 60 μg/mL,from about 45 μg/mL to about 65 μg/mL, from about 50 μg/mL to about 75μg/mL, from about 60 μg/mL to about 240 μg/mL, from about 70 μg/mL toabout 350 μg/mL, from about 80 μg/mL to about 400 μg/mL, from about 90μg/mL to about 450 μg/mL, from about 100 μg/mL to about 500 μg/mL, fromabout 0.01 mg/mL to about 1 mg/mL, from about 0.05 mg/mL to about 2mg/mL, from about 1 mg/mL to about 5 mg/mL, from about 2 mg/mL to about10 mg/mL, from about 4 mg/mL to about 16 mg/mL, from about 5 mg/mL toabout 20 mg/mL, from about 8 mg/mL to about 24 mg/mL, from about 10mg/mL to about 30 mg/mL, from about 12 mg/mL to about 32 mg/mL, fromabout 14 mg/mL to about 34 mg/mL, from about 16 mg/mL to about 36 mg/mL,from about 18 mg/mL to about 38 mg/mL, from about 20 mg/mL to about 40mg/mL, from about 22 mg/mL to about 42 mg/mL, from about 24 mg/mL toabout 44 mg/mL, from about 26 mg/mL to about 46 mg/mL, from about 28mg/mL to about 48 mg/mL, from about 30 mg/mL to about 50 mg/mL, fromabout 35 mg/mL to about 55 mg/mL, from about 40 mg/mL to about 60 mg/mL,from about 45 mg/mL to about 65 mg/mL, from about 50 mg/mL to about 75mg/mL, from about 60 mg/mL to about 240 mg/mL, from about 70 mg/mL toabout 350 mg/mL, from about 80 mg/mL to about 400 mg/mL, from about 90mg/mL to about 450 mg/mL, from about 100 mg/mL to about 500 mg/mL, fromabout 0.01 g/mL to about 1 g/mL, from about 0.05 g/mL to about 2 g/mL,from about 1 g/mL to about 5 g/mL, from about 2 g/mL to about 10 g/mL,from about 4 g/mL to about 16 g/mL, or from about 5 g/mL to about 20g/mL.

Gels and Hydrogels

In some embodiments, SBPs are or are combined with gels or hydrogels. Asused herein, the term “gel” refers to a dispersion of liquid moleculesin a solid medium. Gels in which the dispersed liquid molecules includewater are referred to herein as “hydrogels.” Gels in which the dispersedliquid molecules include an organic phase are referred to herein as“organogels.” The solid medium may include polymer networks.

In some embodiments, SBP gels or hydrogels are prepared with processedsilk. In processed silk gels, polymer networks may include silk fibroin.In some embodiments, gels are prepared with one or more therapeuticagents. In some embodiments, gels include one or more excipients. Theexcipients may be selected from any of those described herein. In someembodiments, excipients may include salts. In some embodiments, theexcipients may include gelling agents. In some embodiments, gels areprepared with one or more therapeutic agents, biological agents,proteins, small molecules, and/or polymers.

Gel preparation may require varying temperatures and incubation timesfor gel polymer networks to form. In some embodiments, processed silkpreparations are heated to 37° C. to prepare gels. In some embodiments,processed silk preparations are incubated for from about 2 hours toabout 36 hours or more to promote gel formation. In some embodiments,gel formation requires mixing with one or more gelling agents orexcipients. Mixing may be carried out under various temperatures andlengths of time to allow gel polymer networks to form. Gel formation mayrequire homogenous dispersion of gelling agents or excipients. In someembodiments, processed silk preparations used to prepare gels includesilk fibroin. Gel formation for processed silk gels may requireincubation at 37° C. for up to 24 hours. Some gels may be stored forlater use or processing. In some embodiments, gels are stored at 4° C.

In some embodiments, processed silk gels include excipient or gellingagent at a concentration of from about 0.01% to about 0.1%, from about0.1% (w/v) to about 1% (w/v), from about 0.5% (w/v) to about 5% (w/v),from about 1% (w/v) to about 10% (w/v), from about 5% (w/v) to about 15%(w/v), from about 10% (w/v) to about 30% (w/v), from about 15% (w/v) toabout 45% (w/v), from about 20% (w/v) to about 55% (w/v), from about 25%(w/v) to about 65% (w/v), from about 30% (w/v) to about 70% (w/v), fromabout 35% (w/v) to about 75% (w/v), from about 40% (w/v) to about 80%(w/v), from about 50% (w/v) to about 85% (w/v), from about 60% (w/v) toabout 90% (w/v), from about 75% (w/v) to about 95% (w/v), from about 90%(w/v) to about 96% (w/v), from about 92% (w/v) to about 98% (w/v), fromabout 95% (w/v) to about 99% (w/v), from about 98% (w/v) to about 99.5%(w/v), or from about 99% (w/v) to about 99.9% (w/v).

In some embodiments, processed silk gels (e.g., hydrogels or organogels)include silk fibroin at a concentration of from about 0.01% to about0.1%, from about 0.1% (w/v) to about 1% (w/v), from about 0.5% (w/v) toabout 5% (w/v), from about 1% (w/v) to about 10% (w/v), from about 5%(w/v) to about 15% (w/v), from about 10% (w/v) to about 30% (w/v), fromabout 15% (w/v) to about 45% (w/v), from about 20% (w/v) to about 55%(w/v), from about 25% (w/v) to about 65% (w/v), from about 30% (w/v) toabout 70% (w/v), from about 35% (w/v) to about 75% (w/v), from about 40%(w/v) to about 80% (w/v), from about 50% (w/v) to about 85% (w/v), fromabout 60% (w/v) to about 90%4 (w/v), from about 75% (w/v) to about 95%(w/v), from about 90% (w/v) to about 96% (w/v), from about 92% (w/v) toabout 98% (w/v), from about 95% (w/v) to about 99% (w/v), from about 98%(w/v) to about 99.5% (w/v), or from about 99% (w/v) to about 99.9%(w/v). Silk fibroin included may be from a silk fibroin preparation withan average silk fibroin molecular weight or range of molecular weightsof from about 3.5 kDa to about 10 kDa, from about 5 kDa to about 20 kDa,from about 10 kDa to about 30 kDa, from about 15 kDa to about 40 kDa,from about 20 kDa to about 50 kDa, from about 25 kDa to about 60 kDa,from about 30 kDa to about 70 kDa, from about 35 kDa to about 80 kDa,from about 40 kDa to about 90 kDa, from about 45 kDa to about 100 kDa,from about 50 kDa to about 110 kDa, from about 55 kDa to about 120 kDa,from about 60 kDa to about 130 kDa, from about 65 kDa to about 140 kDa,from about 70 kDa to about 150 kDa, from about 75 kDa to about 160 kDa,from about 80 kDa to about 170 kDa, from about 85 kDa to about 180 kDa,from about 90 kDa to about 190 kDa, from about 95 kDa to about 200 kDa,from about 100 kDa to about 210 kDa, from about 115 kDa to about 220kDa, from about 125 kDa to about 240 kDa, from about 135 kDa to about260 kDa, from about 145 kDa to about 280 kDa, from about 155 kDa toabout 300 kDa, from about 165 kDa to about 320 kDa, from about 175 kDato about 340 kDa, from about 185 kDa to about 360 kDa, from about 195kDa to about 380 kDa, from about 205 kDa to about 400 kDa, from about215 kDa to about 420 kDa, from about 225 kDa to about 440 kDa, fromabout 235 kDa to about 460 kDa. or from about 245 kDa to about 500 kDa.

Gelling agents may be used to facilitate sol-gel transition. As usedherein, the term “sol-gel transition” refers to the shift of aformulation from a solution to a gel. In some embodiments, the use ofgelling agents may be carried out according to any of such methodsdescribed in International Publication No. WO2017139684, the contents ofwhich are herein incorporated by reference in their entirety. Gellingagents may be water-soluble, waxy solids. In some embodiments, gellingagents may be water-soluble and hygroscopic in nature. In someembodiments, gelling agents may include polar molecules. Gelling agentsmay have net positive, net negative, or net neutral charges at aphysiological pH (e.g., pH of about 7.4). Some gelling agents may beamphipathic. Additional examples of gelling agents include oils (e.g.,castor, corn oil, cottonseed oil, olive oil, peanut oil, peppermint oil,safflower oil, sesame oil, soybean oil, hydrogenated vegetable oil,hydrogenated soybean oil, and medium-chain triglycerides of coconut oiland/or palm seed oil), emulsifiers [e.g., polyoxyl 40 stearate (PEG 1750monosterate), polyoxyl 8 stearate (PEG 400 monosterate), polysorbate 20,polysorbate-SO, or poloxamer], surfactants (e.g., polysorbate,poloxamer, sodium dodecyl sulfate, Triton X100, or tyloxapol), andsuspending agents (e.g., polyvinyl pyrrolidone, polyvinylpyrrolidone-12, polyvinyl pyrrolidone-17, hydroxyethyl cellulose, orcarboxymethyl cellulose).

In some embodiments, gel formation is induced by applying one or more ofthe following to processed silk preparations: ultrasound, sonication,shear forces, temperature change (e.g., heating), addition ofprecipitants, modulation of pH, changes in salt concentration, chemicalcross-linking, chemical modification, seeding with preformed hydrogels,increasing silk fibroin concentration, modulating osmolarity, use ofelectric fields, or exposure to electric currents. In some embodiments,methods of inducing gel formation may include, but are not limited toany of those described in International Publication No. WO2005012606 orUnited States Publication No. US2011/0171239, the contents of each ofwhich are herein incorporated by reference in their entirety.

In some embodiments, processed silk gel preparation may be carried withthe aid of sonication. As used herein, the term “sonication” refers to aprocess of agitation using sound energy. Sonication conducted atfrequencies greater than 20 kHz is referred to as ultrasonication.Sonication may aid in gel formation by dispersing and/or agitatingpolymer components within a solution to foster an arrangement thatfavors polymer network formation. The polymer network may include silkfibroin. In some embodiments, the use of sonication for gel preparationmay be carried out according to any of the methods described in Zhao etal. (2017) Materials Letters 211:110-113 or Mao et al. (2017) ColloidsSurf B Biointerfaces 160:704-714), the contents of each of which areherein incorporated by reference in their entirety.

In some embodiments, processed silk gel formation may be carried outusing shear forces. As used herein, the term “shear forces” refers tounaligned forces that apply pressure to two or more different parts ofan object or medium from different or opposing directions. Shear forcesare distinct from compression forces, which are directed toward eachother. Shear forces may be applied during processed silk gel preparationusing a syringe, tubing, needle, or other apparatus capable ofincreasing shear forces. Processed silk preparation may be pushedthrough a syringe, tubing, needle, or other apparatus to generate shearforces. The use of shear forces in gel formation may include any ofthose described in United States Publication No. US2011/0171239, thecontents of which are herein incorporated by reference in theirentirety.

In some embodiments, changes in temperature may be used to aid inprocessed silk gel formation. Changes in temperature may be used todisperse or align polymer components in an arrangement that promotes gelpolymer network formation. The polymer components may include silkfibroin. In some embodiments, gel formation may be carried out byraising or lowering the temperature of a processed silk preparation tofrom about 0° C. to about 5° C., from about 2° C. to about 6° C., fromabout 4° C. to about 12° C., from about 8° C. to about 16° C., fromabout 10° C. to about 26° C., from about 15° C. to about 28° C., fromabout 20° C. to about 32° C., from about 25° C. to about 34° C., fromabout 30° C. to about 45° C., from about 35° C. to about 55° C., fromabout 37° C. to about 65° C., from about 40° C. to about 75° C., fromabout 50° C. to about 100° C. from about 60° C. to about 120° C., fromabout 70° C. to about 140° C., from about 80° C. to about 160° C., orfrom about 100° C. to about 300° C. In some embodiments, one or moreexcipients or gelling agents may be included to lower the temperaturenecessary for gel formation to occur. Such embodiments may be employedto protect temperature-sensitive components embedded within gels. Insome embodiments, gel formation is carried out at 4° C. Glycerol,polyethylene glycol (PEG), and/or polymers of PEG (e.g., PEG400) may beincluded in processed silk preparations as excipients to lower thetemperature necessary to form a gel. The gel may be a silk fibroin gel.Excipient concentration may be about 30% (w/v). Silk fibroinconcentration may be from about 2% to about 30%.

In some embodiments, gel formation is carried out by applying anelectric current, also referred to as “electrogelation.” Electrogelationmay be carried out according to any of the methods presented inInternational Publication No. WO2010036992, the contents of which areherein incorporated by reference in their entirety. In some embodiments,a reverse voltage may be applied to reverse gel formation and regeneratea processed silk solution.

In some embodiments, gel formation is carried out by modulating the pHof processed silk preparations. Gel formation through pH modulation maybe carried out according to the methods described in InternationalPublication No. WO2005012606, United States Publication No.US2011/0171239, and Dubey et al. (2017) Materials Chemistry and Physics203:9-16, the contents of each of which are herein incorporated byreference in their entirety.

In some embodiments, gel formation may be carried out in associationwith modulating the osmolarity of a processed silk preparation. As usedherein, the term “osmolarity” or “osmotic concentration” refers to thenumber of osmoles of solute in solution on a per liter basis (Osm/L).Unlike molarity, which is a measure of the number of moles solute perliter of solvent (M), osmolarity factors in the effect of ions onosmotic pressure. For example, a 1 M solution of NaCl would have anosmolarity of 2 Osm/L while a 1 M solution of MgCl₂ would have anosmolarity of 3 Osm/L. Hypo- or hyper-osmotic formulations can lead tolocal tissue damage and reduced biocompatibility. In some embodiments,the osmolarity of processed silk gels is modulated by controlling thetype, molecular weight, and/or concentration of excipients included.Osmolarity may be modulated by varying the concentration and/ormolecular weight of salts used in processed silk preparations. In someembodiments, osmolarity is reduced by using lower molecular weightgelling agents. For example, 4 kDa PEG may be used in place of PEG400.The use of Poloxamer 188 at 10% (w/v) may reduce osmolarity incomparison to lower molecular weight species such as glycerol. In someembodiments, sodium chloride may be added to increase osmolarity. Insome embodiments, osmolarity is adjusted to fall between 280 and 320mOsm/L.

In some embodiments, gel formation may be carried out through seeding.As used herein when referring to gel formation. “seeding” refers to aprocess of inducing gel formation using a small amount of pre-formedgel. Seeding may promote gel formation by encouraging polymer networkformation to build off of the pre-formed gel introduced. In someembodiments the gel includes silk fibroin. Seeding with a pre-formedsilk fibroin hydrogel may be used to promote transition of a silkfibroin solution into a silk fibroin gel. In some embodiments, seedingreduces the need for gelling agents and/or excipients to form gels.

In some embodiments, gel formation may be carried out using chemicalcross-linking. As used herein, the term “chemical cross-linking” refersto a process of forming covalent bonds between chemical groups fromdifferent molecules or between chemical groups present on differentparts of the same molecule. In some embodiments, chemical cross-linkingmay be carried out by contacting processed silk preparations withethanol. Such methods may be carried out according to those described inShi et a. (2017) Advanced Material 29(29):1701089, the contents of whichare herein incorporated by reference in their entirety. In someembodiments, cross-linking may be carried out using enzymes. Methods ofenzyme cross-linking using horse radish peroxidase may include any ofthose described in McGill et a (2017) Acta Biomaterialia 63:76-84 or Guoet a. (2017) Biomaterials 145:44-55, the contents of each of which areherein incorporated by reference in their entirety. In some embodiments,chemical cross-linking may be photo-initiated, as disclosed inInternational Publication No. WO2017123383 and in Zhang et al. (2017)Fibers and Polymers 18(10):1831-1840, the contents of each of which areherein incorporated by reference in their entirety.

In some embodiments, other chemical modifications may be used duringprocessed silk gel preparation. Some chemical modifications may be usedto induce silk fibroin p-sheet conformations. In some embodiments, thisprocess involves contact with a chemical. Chemicals may include, but arenot limited to, ethanol. In some embodiments, silk fibroin may bechemically crosslinked with other materials during gel preparation. Suchmaterials may include other peptides (e.g., see Guo et al. (2017)Biomaterials 145:44-55, the contents of which are herein incorporated byreference in their entirety). In some embodiments, processed silk gelsare prepared by formation of internal chemical cross-links. Thesecrosslinks may be dityrosine crosslinks (e.g., see InternationalPublication No. WO2017123383, the contents of which are hereinincorporated by reference in their entirety). In some embodiments,photosensitive materials may be used to promote chemical modifications.Such materials may include riboflavin (e.g., see InternationalPublication No. WO2017123383). In some embodiments, processed silk gelsmay be functionalized with particles. These particles may bemicrospheres and/or nanospheres (e.g., see Ciocci et al. (2017) Int JBiol Macromol S0141-8130(17):32839-8, the contents of which are hereinincorporated by reference in their entirety).

Particles

In some embodiments, SBPs may be particles. As used herein, the term“particle” refers to a minute portion of a substance. SBP particles mayinclude particles of processed silk. Processed silk particles mayinclude silk fibroin particles. Silk fibroin particles may be tinyclusters of silk fibroin or they may be arranged as more orderedstructures. Particles may vary in size. Processed silk particles may bevisible or may be too tiny to view easily with the naked eye. Particleswith a width of from about 0.1 μm to about 100 μm are referred to hereinas “microparticles.” Particles with a width of about 100 nm or less arereferred to herein as “nanoparticles.” Microparticles and nanoparticlesthat are spherical in shape are termed microspheres and nanospheres,respectively. Processed silk particle preparations may include particleswith uniform width or with ranges of widths. In some embodiments,processed silk particle preparations include average particle widths ofor ranges of particle widths of from about 10 nm to about 25 nm, fromabout 20 nm to about 50 nm, from about 30 nm to about 75 nm, from about40 nm to about 80 nm, from about 50 nm to about 100 nm, from about 0.05μm to about 10 μm, from about 1 μm to about 20 μm, from about 2 μm toabout 30 μm, from about 5 μm to about 40 μm, from about 10 μm to about50 μm, from about 20 μm to about 60 μm, from about 30 μm to about 70 μm,from about 40 μm to about 80 μm, from about 50 μm to about 90 μm, fromabout 0.05 mm to about 2 mm, from about 0.1 mm to about 3 mm, from about0.2 mm to about 4 mm, from about 0.5 mm to about 5 mm, from about 1 mmto about 6 mm, from about 2 mm to about 7 mm, from about 5 mm to about10 mm, from about 10 nm to about 100 μm, from about 10 μm to about 10mm, from about 50 nm to about 500 μm, from about 50 μm to about 5 mm,from about 100 nm to about 10 mm, or from about 1 μm to about 10 mm. Insome embodiments, processed silk particle preparations include averageparticle widths of at least 10 nm, at least 100 nm, at least 0.5 μm, atleast 1 μm at least 10 μm, at least 100 μm, at least 500 μm, at least 1mm, or at least 10 mm.

Processed silk particles may be formed through spraying of a processedsilk preparation. In some embodiments, electrospraying is used.Electrospraying may be carried out using a coaxial electrosprayapparatus (e.g., see Cao et 71. (2017) Scientific Reports 7:11913, thecontents of which are herein incorporated by reference in theirentirety). In some embodiments, silk fibroin microspheres or nanospheresmay be obtained by electrospraying a silk fibroin preparation into acollector and flash freezing the sprayed particles (e.g., see UnitedStates Publication No. US2017/0333351, the contents of which are hereinincorporated by reference in their entirety). The flash frozen silkfibroin particles may then be lyophilized. In some embodiments,processed silk particles may be prepared using centrifugal washing,followed by lyophilization, as taught in United States Publication No.US2017/0340575, the contents of which are herein incorporated byreference in their entirety. In some embodiments, processed silkmicrospheres may be formed through the use of a microfluidic device(e.g., see Sun et al. (2017) Journal of Materials Chemistry B5:8770-8779, the contents of which are herein incorporated by referencein their entirety). In some embodiments, microspheres are formed viacoagulation in a methanol bath, as taught in European Patent No.EP3242967, the contents of which are herein incorporated by reference intheir entirety.

Scaffolds

In some embodiments, SBPs include scaffolds. As used herein, a“scaffold” refers to a framework used for support. SBP scaffolds mayinclude scaffolds formed using processed silk frameworks. Processed silkmay include a polymeric network that provides a framework to support avariety of materials related to a variety of applications. Suchapplication may include, but are not limited to, therapeuticapplications. In some embodiments, processed silk scaffolds includepolymeric networks that include silk fibroin. In some embodiments,processed silk scaffolds include one or more of silk fibers, nanofibers,mats, films, foams, membranes, rods, tubes, gels, hydrogels,microspheres, nanospheres, solutions, patches, grafts, and powders. Insome embodiments, processed silk scaffolds include other agents. Suchagents may include, but are not limited to, therapeutic agents (e.g.,ocular therapeutic agents, for example, NSAIDS).

In some embodiments, processed silk scaffolds are prepared by casting aprocessed silk preparation into a mold, and allowing the preparation tosolidify to obtain the desired shape. Any mold shape may be used. Insome embodiments, injection molding machines are used. Molding may beperformed at various temperatures needed to facilitate filling of moldsand solidification into final molded form. In some embodiments, moldingis performed at room temperature. In other embodiments, the molding isperformed at 160° C. In some embodiments, molding is carried outaccording to the methods described in International Publication No.WO2017179069, Thai et al. J Biomed Mater (2017) 13(1):015009, or Chen etal. (2017) PLoS One 12(11):e0187880, the contents of each of which areherein incorporated by reference in their entirety.

In some embodiments, processed silk scaffolds are prepared by coating ascaffold formed from non-silk materials with a processed silkpreparation. The processed silk may include silk fibroin. The non-silkmaterials may include, but are not limited to, natural or syntheticpolymers, fibers, nanofibers, mats, films, foams, membranes, rods,tubes, gels, hydrogels, microspheres, nanospheres, nanoparticles,particles, solutions, patches, and/or grafts. Methods of coating ascaffold with a processed silk preparation are taught in Ai et at (2017)International Journal of Nanomedicine 12:7737-7750 and Jiang et al.(2017) J Biomater Sci Polym Ed 15:1-36, the contents of each of whichare herein incorporated by reference in their entirety.

Devices

In some embodiments, SBPs may be devices or may be included as devicecomponents. As used herein, the term “device” refers to any articleconstructed or modified to suit a particular purpose. Devices may bedesigned for a variety of purposes, including, but not limited to,therapeutic applications. In some embodiments, SBPs are embedded orincorporated into devices. Some devices include SBPs as coatings orlubricants. In some embodiments, devices include implants, medicaldevices, or surgical devices. Additional devices are described herein.

Ocular SBPs

SBPs described herein may include ocular SBPs. As used herein, the term“ocular SBP” refers to an SBP used in any application related to theeye. Ocular SBPs may be used in therapeutic applications. Suchtherapeutic applications may include treating or otherwise addressingone or more ocular indications.

Ocular SBPs may be prepared in a variety of formats. Some ocular SBPsare prepared in the shape of a rod. Some ocular SBPs may be in the formof a lyophilized powder. Some ocular SBPs are in the form of a hydrogel.Other ocular SBPs may be in the form of a solution. Ocular SBPs mayinclude ocular therapeutic agents. The ocular therapeutic agents mayinclude any of those described herein. In some embodiments, oculartherapeutic agents include one or more of processed silk, biologicalagents, small molecules, proteins, NSADs, and VEGF-related agents.Ocular therapeutic agent proteins may include, but are not limited to,lysozyme, bovine serum albumin (BSA), bevacizumab, or VEGF-relatedagents. NSAIDs may include, but are not limited to, aspirin, carprofen,celecoxib, deracoxib, diclofenac, diflunisal, etodolac, fenoprofen,firocoxib, flurbirofen, ibuprofen, indomethacin, ketoprofen, ketorolac,mefenamic acid, meloxicam, nabumetone, naproxen, oxaprozin, piroxicam,robenacoxib, salsalate, sulindac, and tolmetin. In some embodiments, theSBPs stabilize ocular therapeutic agents included. Ocular SBPs mayinclude ocular therapeutic agent concentrations [expressed as percentageof ocular therapeutic agent weight contributing to total SBP weight] offrom about 0.1% to about 98% (w/w). For example, SBPs may include oculartherapeutic agents at a concentration of from about 0.01% (w/w) to about1% (w/w), from about 0.05% (w/w) to about 2% (w/w), from about 1% (w/w)to about 5% (w/w), from about 2% (w/w) to about 10% (w/w), from about 4%(w/w) to about 16% (w/w), from about 5% (w/w) to about 20% (w/w), fromabout 5% (w/w) to about 85% (w/w), from about 8% (w/w) to about 24%(w/w), from about 10% (w/w) to about 30% (w/w), from about 12% (w/v) toabout 32% (w/w), from about 14% (w/v) to about 34% (w/v), from about 15%(w/w) to about 95% (w/w), from about 16% (w/w) to about 36% (w/w), fromabout 18% (w/w) to about 38% (w/w), from about 20% (w/w) to about 40%(w/w), from about 22% (w/w) to about 42% (w/w), from about 24% (w/w) toabout 44% (w/w), from about 26% (w/w) to about 46% (w/w), from about 28%(w/w) to about 48% (w/w), from about 30% (w/w) to about 50% (w/w), fromabout 35% (w/v) to about 55% (w/), from about 40% (w/w) to about 60%(w/w), from about 45% (w/w) to about 65% (w/w), from about 50% (w/w) toabout 70% (w/w), from about 55% (w/v) to about 75% (w/w), from about 60%(w/w) to about 80% (w/w), from about 65% (w/w) to about 85% (w/w), fromabout 70% (w/w) to about 90% (w/w), from about 75% (w/w) to about 95%(w/w), from about 80% (w/w) to about 96% (w/w), from about 85% (w/w) toabout 97% (w/w), from about 90% (w/w) to about 98% (w/w), from about 95%(w/w) to about 99% (w/w), from about 96% (w/w) to about 99.2% (w/w), orfrom about 97% (w/w) to about 98% (w/w). The SBPs may include a ratio ofocular therapeutic agent (by weight, volume, or concentration) toprocessed silk (by weight, volume, or concentration) of from about0.001:1 to about 1:1, from about 0.005:1 to about 5:1, from about 0.01:1to about 1:1, from about 0.01:1 to about 4.2:1, from about 0.01:1 toabout 10:1, from about 0.02:1 to about 20:1, from about 0.03:1 to about30:1, from about 0.04:1 to about 40:1, from about 0.05:1 to about 50:1,from about 0.06:1 to about 60:1, from about 0.07:1 to about 70:1, fromabout 0.08:1 to about 80:1, from about 0.09:1 to about 90:1, from about0.1:1 to about 100:1, from about 0.2:1 to about 150:1, from about 0.3:1to about 200:1, from about 0.3:1 to about 4.2:1, from about 0.4:1 toabout 250:1, from about 0.5:1 to about 300:1, from about 0.6:1 to about350:1, from about 0.7:1 to about 400:1, from about 0.8:1 to about 450:1,from about 0.9:1 to about 500:1, from about 1:1 to about 4.2:1, fromabout 1:1 to about 550:1, from about 2:1 to about 600:1, from about 3:1to about 650:1, from about 4:1 to about 700:1, from about 5:1 to about750:1, from about 6:1 to about 800:1, from about 7:1 to about 850:1,from about 8:1 to about 900:1, from about 9:1 to about 950:1, from about10:1 to about 960:1, from about 50:1 to about 970:1, from about 100:1 toabout 980:1, from about 200:1 to about 990:1, or from about 500:1 toabout 1000:1. The processed silk may be or include silk fibroin.

Ocular SBPs may include one or more excipients. The excipients mayinclude any of those described herein. In some embodiments, theexcipients include one or more of lactose, sorbitol, sucrose, mannitol,lactose USP, Starch 1500, microcrystalline cellulose, Avicel, phosphatesalts, sodium chloride, potassium phosphate monobasic, potassiumphosphate dibasic, sodium phosphate dibasic, sodium phosphate monobasic,polysorbate 80, phosphate buffer, phosphate buffered saline, sodiumhydroxide, hydrochloric acid, dibasic calcium phosphate dehydrate,tartaric acid, citric acid, fumaric acid, succinic acid, malic acid,polyvinylpyrrolidone, copolymers of vinylpyrrolidone and vinylacetate,hydroxypropylcellulose, hydroxyethylcellulose,hydroxypropylmethylcellulose, polyvinyl alcohol, polyethylene glycol,acacia, and sodium carboxymethylcellulose. SBPs may include at least oneexcipient at a concentration of from about 1% to about 20% (w/w). Insome embodiments, SBPs include at least one excipient at a concentrationof from about 0.01% to about 1%, from about 0.05% to about 2%, fromabout 1% to about 5%, from about 2% to about 10%, from about 3% to about15%, from about 4% to about 20%, from about 5% to about 25%, from about6% to about 30%, from about 7% to about 35%, from about 8% to about 40%,from about 9% to about 45%, from about 10% to about 50%, from about 12%to about 55%, from about 14% to about 60%, from about 16% to about 65%,from about 18% to about 70%, from about 20% to about 75%, from about 22%to about 80%, from about 24% to about 85%, from about 26% to about 90%,from about 28% to about 95%, from about 30% to about 96%, from about 32%to about 97%, from about 34% to about 98%, from about 36% to about98.5%, from about 38% to about 99%, from about 40% to about 99.5%, fromabout 42% to about 99.6%, from about 44% to about 99.7%, from about 46%to about 99.8%, or from about 50% to about 99.9%. SBPs may include aratio of ocular therapeutic agent (by weight, volume, or concentration)to at least one excipient (by weight, volume, or concentration) of fromabout 0.001:1 to about 1:1, from about 0.005:1 to about 5:1, from about0.01:1 to about 0.5:1, from about 0.01:1 to about 10:1, from about0.02:1 to about 20:1, from about 0.03:1 to about 30:1, from about 0.04:1to about 40:1, from about 0.05:1 to about 50:1, from about 0.06:1 toabout 60:1, from about 0.07:1 to about 70:1, from about 0.08:1 to about80:1, from about 0.09:1 to about 90:1, from about 0.1:1 to about 100:1,from about 0.2:1 to about 150:1, from about 0.3:1 to about 200:1, fromabout 0.4:1 to about 250:1, from about 0.5:1 to about 300:1, from about0.6:1 to about 350:1, from about 0.7:1 to about 400:1, from about 0.8:1to about 450:1, from about 0.9:1 to about 500:1, from about 1:1 to about550:1, from about 2:1 to about 600:1, from about 3:1 to about 650:1,from about 4:1 to about 700:1, from about 5:1 to about 750:1, from about6:1 to about 800:1, from about 7:1 to about 850:1, from about 8:1 toabout 900:1, from about 9:1 to about 950:1, from about 10:1 to about960:1, from about 50:1 to about 970:1, from about 100:1 to about 980:1,from about 200:1 to about 990:1, or from about 500:1 to about 1000:1. Insome embodiments, ocular SBPs contain trace amounts of excipient. Insome embodiments, the excipient is phosphate buffer or phosphatebuffered saline.

Ocular SBPs may have a density of from about 0.01 mg/mL to about 1mg/mL, from about 0.05 mg/mL to about 2 mg/mL, from about 1 mg/mL toabout 5 mg/mL, from about 2 mg/mL to about 10 mg/mL, from about 4 mg/mLto about 16 mg/mL, from about 5 mg/mL to about 20 mg/mL, from about 8mg/mL to about 24 mg/mL, from about 10 mg/mL to about 30 mg/mL, fromabout 12 mg/mL to about 32 mg/mL, from about 14 mg/mL to about 34 mg/mL,from about 16 mg/mL to about 36 mg/mL, from about 18 mg/mL to about 38mg/mL, from about 20 mg/mL to about 40 mg/mL, from about 22 mg/mL toabout 42 mg/mL, from about 24 mg/mL to about 44 mg/mL, from about 26mg/mL to about 46 mg/mL, from about 28 mg/mL to about 48 mg/mL, fromabout 30 mg/mL to about 50 mg/mL, from about 35 mg/mL to about 55 mg/mL,from about 40 mg/mL to about 60 mg/mL, from about 45 mg/mL to about 65mg/mL, from about 50 mg/mL to about 75 mg/mL, from about 60 mg/mL toabout 240 mg/mL, from about 70 mg/mL to about 350 mg/mL, from about 80mg/mL to about 400 mg/mL, from about 90 mg/mL to about 450 mg/mL, fromabout 100 mg/mL to about 500 mg/mL, from about 0.01 g/mL to about 1g/mL, from about 0.05 g/mL to about 2 g/mL, from about 0.7 g/mL to about1.4 g/mL, from about 1 g/mL to about 5 g/mL, from about 2 g/mL to about10 g/mL, from about 4 g/mL to about 16 g/mL, from about 5 g/mL to about20 g/mL, from about 8 g/mL to about 24 g/mL, from about 10 g/mL to about30 g/mL, from about 12 g/mL to about 32 g/mL, from about 14 g/mL toabout 34 g/mL, from about 16 g/mL to about 36 g/mL, from about 18 g/mLto about 38 g/mL, from about 20 g/mL to about 40 g/mL, from about 22g/mL to about 42 g/mL, from about 24 g/mL to about 44 g/mL, from about26 g/mL to about 46 g/mL, from about 28 g/mL to about 48 g/mL, fromabout 30 g/mL to about 50 g/mL, from about 35 g/mL to about 55 g/mL,from about 40 g/mL to about 60 g/mL, from about 45 g/mL to about 65g/mL, from about 50 g/mL to about 75 g/mL, from about 60 g/mL to about240 g/mL, from about 70 g/mL to about 350 g/mL, from about 80 g/mL toabout 400 g/mL, from about 90 g/mL to about 450 g/mL, or from about 100g/mL to about 500 g/mL.

Ocular SBPs may be in the shape of a rod. Such SBPs may include adiameter of from about 0.05 μm to about 10 μm, from about 1 μm to about20 μm, from about 2 μm to about 30 μm, from about 5 μm to about 40 μm,from about 10 μm to about 50 μm, from about 20 μm to about 60 μm, fromabout 30 μm to about 70 μm, from about 40 μm to about 80 μm, from about50 μm to about 90 μm, from about 45 μm to about 100 μm, from about 50 μmto about 110 μm, from about 55 μm to about 120 μm, from about 60 μm toabout 130 μm, from about 65 μm to about 140 μm, from about 70 μm toabout 150 μm, from about 75 μm to about 160 μm, from about 80 μm toabout 170 μm, from about 85 μm to about 180 μm, from about 90 μm toabout 190 μm, from about 95 μm to about 200 μm, from about 100 μm toabout 210 μm, from about 115 μm to about 220 μm, from about 125 μm toabout 240 μm, from about 135 μm to about 260 μm, from about 145 μm toabout 280 μm, from about 155 μm to about 300 μm, from about 165 μm toabout 320 μm, from about 175 μm to about 340 μm, from about 185 μm toabout 360 μm, from about 195 μm to about 380 μm, from about 205 μm toabout 400 μm, from about 215 μm to about 420 μm, from about 225 μm toabout 440 μm, from about 235 μm to about 460 μm, from about 245 μm toabout 500 μm, from about 0.05 mm to about 2 mm, from about 0.1 mm toabout 1.5 mm, from about 0.1 mm to about 3 mm, from about 0.2 mm toabout 4 mm, from about 0.3 mm to about 1.2 mm, from about 0.5 mm toabout 5 mm, from about 1 mm to about 6 mm, from about 2 mm to about 7mm, or from about 5 mm to about 10 mm. SBP rods may have a length offrom about 0.05 mm to about 2 mm, from about 0.1 mm to about 3 mm, fromabout 0.2 mm to about 4 mm, from about 0.3 mm to about 1.2 mm, fromabout 0.5 mm to about 5 mm, from about 1 mm to about 6 mm, from about 2mm to about 7 mm, from about 5 mm to about 10 mm, from about 8 mm toabout 12 mm, from about 10 mm to about 15 mm, from about 12 mm to about18 mm, from about 15 mm to about 25 mm, or from about 20 mm to about 30mm.

Ocular SBPs may be hydrogels. Such SBPs may include at least oneexcipient selected from one or more of sorbitol, triethylamine,2-pyrrolidone, alpha-cyclodextrin, benzyl alcohol, beta-cyclodextrin,dimethyl sulfoxide, dimethylacetamide (DMA), dimethylformamide, ethanol,gamma-cyclodextrin, glycerol, glycerol formal, hydroxypropylbeta-cyclodextrin, kolliphor 124, kolliphor 181, kolliphor 188,kolliphor 407, kolliphor EL (cremaphor EL), cremaphor RH 40, cremophorRH 60, dalpha-tocopherol, PEG 1000 succinate, polysorbate 20,polysorbate 80, solutol HS 15, sorbitan monooleate, poloxamer-407,poloxamer-188, Labrafil M-1944CS, Labrafil M-2125CS, Labrasol, Gellucire44/14. Softigen 767, mono- and di-fatty acid esters of PEG 300, PEG 400,or PEG 1750, kolliphor RH60, N-methyl-2-pyrrolidone, castor oil, cornoil, cottonseed oil, olive oil, peanut oil, peppermint oil, saffloweroil, sesame oil, soybean oil, hydrogenated vegetable oils, hydrogenatedsoybean oil, medium chain triglycerides of coconut oil, medium chaintriglycerides of palm seed oil, beeswax, d-alpha-tocopherol, oleic acid,medium-chain mono-glycerides, medium-chain di-glycerides,alpha-cyclodextrin, betacyclodextrin, hydroxypropyl-beta-cyclodextrin,sulfo-butylether-beta-cyclodextrin, hydrogenated soyphosphatidylcholine, distearoylphosphatidylglycerol,L-alphadimyristoylphosphatidylcholine,L-alpha-dimyristoylphosphatidylglycerol, PEG 300, PEG 300caprylic/capric glycerides (Softigen 767), PEG 300 linoleic glycerides(Labrafil M-2125CS). PEG 300 oleic glycerides (Labrafil M-1944CS), PEG400, PEG 400 caprylic/capric glycerides (Labrasol), polyoxyl 40 stearate(PEG 1750 monosterate), polyoxyl 8 stearate (PEG 400 monosterate),polysorbate 20, polysorbate 80, polyvinyl pyrrolidone, propylenecarbonate, propylene glycol, solutol HS15, sorbitan monooleate (Span20), sulfobutylether-beta-cyclodextrin, transcutol, triacetin,1-dodecylazacyclo-heptan-2-one, caprolactam, castor oil, cottonseed oil,ethyl acetate, medium chain triglycerides, methyl acetate, oleic acid,safflower oil, sesame oil, soybean oil, tetrahydrofuran, glycerin, andPEG 4 kDa.

The SBPs may have an osmotic concentration of from about 1 mOsm to about10 mOsm, from about 2 mOsm to about 20 mOsm, from about 3 mOsm to about30 mOsm, from about 4 mOsm to about 40 mOsm, from about 5 mOsm to about50 mOsm, from about 6 mOsm to about 60 mOsm, from about 7 mOsm to about70 mOsm, from about 8 mOsm to about 80 mOsm, from about 9 mOsm to about90 mOsm, from about 10 mOsm to about 100 mOsm, from about 15 mOsm toabout 150 mOsm, from about 25 mOsm to about 200 mOsm, from about 35 mOsmto about 250 mOsm, from about 45 mOsm to about 300 mOsm, from about 55mOsm to about 350 mOsm, from about 65 mOsm to about 400 mOsm, from about75 mOsm to about 450 mOsm, from about 85 mOsm to about 500 mOsm, fromabout 125 mOsm to about 600 mOsm, from about 175 mOsm to about 700 mOsm,from about 225 mOsm to about 800 mOsm, from about 275 mOsm to about 285mOsm, from about 280 mOsm to about 900 mOsm, or from about 325 mOsm toabout 1000 mOsm.

The SBPs may have an osmolarity of from about 1 mOsm/L to about 10mOsm/L, from about 2 mOsm/L to about 20 mOsm/L, from about 3 mOsm/L toabout 30 mOsm/L, from about 4 mOsm/L to about 40 mOsm/L, from about 5mOsm/L to about 50 mOsm/L, from about 6 mOsm/L to about 60 mOsm/L, fromabout 7 mOsm/L to about 70 mOsm/L, from about 8 mOsm/L to about 80mOsm/L, from about 9 mOsm/L to about 90 mOsm/L, from about 10 mOsm/L toabout 100 mOsm/L, from about 15 mOsm/L to about 150 mOsm/L, from about25 mOsm/L to about 200 mOsm/L, from about 35 mOsm/L to about 250 mOsm/L,from about 45 mOsm/L to about 300 mOsm/L, from about 55 mOsm/L to about350 mOsm/L, from about 65 mOsm/L to about 400 mOsm/L, from about 75mOsm/L to about 450 mOsm/L, from about 85 mOsm/L to about 500 mOsm/L,from about 125 mOsm/L to about 600 mOsm/L, from about 175 mOsm/L toabout 700 mOsm/L, from about 225 mOsm/L to about 800 mOsm/L, from about275 mOsm/L to about 285 mOsm/L, from about 280 mOsm/L to about 900mOsm/L, or from about 325 mOsm/L to about 1000 mOsm/L.

In some embodiment, the SBP formulation has an osmolarity from about280-320 mOsm/L.

In some embodiment, the SBP formulation has an osmolarity from about290-320 mOsm/L.

In some embodiment, the SBP formulation has an osmolarity of 280 mOsm/L.

In some embodiment, the SBP formulation has an osmolarity of 290 mOsm/L.Ocular SBPs may have a pH from about 3 to about 10. In some embodiments,the pH is from about 3 to about 6, from about 6 to about 8, or fromabout 8 to about 10. In some embodiments, the pH of the SBP is about7.4.

Ocular SBPs may include silk fibroin. The silk fibroin may be includedat a concentration (w/w or w/v) of 0.01% to about 1%, from about 0.05%to about 2%, from about 0.1% to about 30%, from about 1% to about 5%,from about 2% to about 10%, from about 3% to about 15%, from about 4% toabout 20%, from about 5% to about 25%, from about 6% to about 30%, fromabout 7% to about 35%, from about 8% to about 40%, from about 9% toabout 45%, from about 10% to about 50%, from about 12% to about 55%,from about 14% to about 60%, from about 16% to about 65%, from about 18%to about 70%, from about 20% to about 75%, from about 22% to about 80%,from about 24% to about 85%, from about 26% to about 90%, from about 28%to about 95%, from about 30% to about 96%, from about 32% to about 97%,from about 34% to about 98%, from about 36% to about 98.5%, from about38% to about 99%, from about 40% to about 99.5%, from about 42% to about99.6%, from about 44% to about 99.7%, from about 46% to about 99.8%, orfrom about 50% to about 99.9%. SBPs may include a ratio of silk fibroin(by weight, volume, or concentration) to at least one excipient and/orocular therapeutic agent (by weight, volume, or concentration) of fromabout 0.001:1 to about 1:1, from about 0.005:1 to about 5:1, from about0.01:1 to about 0.5:1, from about 0.01:1 to about 10:1, from about0.02:1 to about 20:1, from about 0.03:1 to about 30:1, from about 0.04:1to about 40:1, from about 0.05:1 to about 50:1, from about 0.06:1 toabout 60:1, from about 0.07:1 to about 70:1, from about 0.08:1 to about80:1, from about 0.09:1 to about 90:1, from about 0.1:1 to about 100:1,from about 0.2:1 to about 150:1, from about 0.3:1 to about 200:1, fromabout 0.4:1 to about 250:1, from about 0.5:1 to about 300:1, from about0.6:1 to about 350:1, from about 0.7:1 to about 400:1, from about 0.8:1to about 450:1, from about 0.9:1 to about 500:1, from about 1:1 to about550:1, from about 2:1 to about 600:1, from about 3:1 to about 650:1,from about 4:1 to about 700:1, from about 5:1 to about 750:1, from about6:1 to about 800:1, from about 7:1 to about 850:1, from about 8:1 toabout 900:1, from about 9:1 to about 950:1, from about 10:1 to about960:1, from about 50:1 to about 970:1, from about 100:1 to about 980:1,from about 200:1 to about 990:1, or from about 500:1 to about 1000:1. Insome embodiments, ocular SBPs contain trace amounts of excipient. Insome embodiments, the excipient is phosphate buffer or phosphatebuffered saline.

SBP viscosity may be modulated by modulating silk fibroin molecularweight and/or concentration. In some embodiments, SBP viscosityincreases with increasing levels of silk fibroin. In some embodiments,SBP viscosity may be tuned by the molecular weight of processed silk, asdefined by the minute boil. In some embodiments, the viscosity of an SBPis proportional to the molecular weight of the processed silk. In someembodiments, the viscosity of an SBP is from about 7 Pa s⁻¹ to about 170Pa s⁻¹. In some embodiments, the viscosity of an SBP is from about 5 Pas⁻¹ to about 200 Pa s⁻¹. In some embodiments, the viscosity of an SBP isfrom about 5 Pa s⁻¹ to about 25 Pa s⁻¹, from about 25 Pa s⁻¹ to about 50Pa s⁻¹, from about 50 Pa s⁻¹ to about 75 Pa s⁻¹, from about 75 Pa s⁻¹ toabout 100 Pa s⁻¹, from about 100 Pa s⁻¹ to about 125 Pa s⁻¹, from about125 Pa s⁻¹ to about 150 Pa s⁻¹, from about 150 Pa s⁻¹ to about 175 Pas⁻¹, or from about 175 Pa s⁻¹ to about 200 Pa s⁻¹. In some embodiments,the stiffness of the SBP may be tuned with the molecular weight of theprocessed silk. In some embodiments, a preparation of an SBP fromprocessed silk with a longer boiling time may enhance the stiffness ofthe SBP. In some embodiments, the viscosity and/or the stiffness of theSBP may be modulated without altering the release kinetics of atherapeutic agent from the SBP.

In some embodiments, ocular SBPs are formulated for intraocularadministration. In some embodiments, ocular SBPs are formulated for oneor more of intravitreal administration, intraretinal administration,intracomeal administration, intrascleral administration, punctaladministration, administration to the anterior sub-Tenon's,suprachoroidal administration, administration to the posteriorsub-Tenon's, subretinal administration, administration to the fornix,administration to the lens, administration to the anterior segment,administration to the posterior segment, macular administration, andintra-aqueous humor administration. Ocular SBPs may be biocompatible,well tolerated, and/or non-immunogenic.

In some embodiments, the present disclosure provides methods of treatingsubjects by contacting them with ocular SBPs. The subjects may have, maybe suspected of having, and/or may be at risk for developing one or moreocular indications. Such ocular indications may include any of thosedescribed herein. In some embodiments, ocular indications includeinflammation. In some embodiments, ocular indications include one ormore of an infection, refractive errors, macular edema, age relatedmacular degeneration, cystoid macular edema, cataracts, diabeticretinopathy (proliferative and non-proliferative), glaucoma, amblyopia,strabismus, color blindness, cytomegalovirus retinitis, keratoconus,diabetic macular edema (proliferative and non-proliferative), lowvision, ocular hypertension, retinal detachment, eyelid twitching,inflammation, uveitis, bulging eyes, dry eye disease, floaters,xerophthalmia, diplopia, Graves' disease, night blindness, eye strain,red eyes, nystagmus, presbyopia, excess tearing, retinal disorder,conjunctivitis, cancer, comeal ulcer, comeal abrasion, snow blindness,scleritis, keratitis, Thygeson's superficial punctate keratopathy,comeal neovascularization, Fuch's dystrophy, keratoconjunctivitis sicca,iritis, chorioretinal inflammation (e.g. chorioretinitis, choroiditis,retinitis, retinochoroiditis, pars planitis, Harada's disease, aniridia,macular scars, solar retinopathy, choroidal degeneration, choroidaldystrophy, choroideremia, gyrate atrophy, choroidal hemorrhage,choroidal detachment, retinoschisis, hypertensive retinopathy, Bull'seye maculopathy, epiretinal membrane, peripheral retinal degeneration,hereditary retinal dystrophy, retinitis pigmentosa, retinal hemorrhage,retinal vein occlusion, and separation of retinal layers.

In some embodiments, the ocular indication is DME. In some embodiments,the ocular indication is diabetic retinopathy. In some embodiments, theocular indication is non-proliferative diabetic retinopathy.

In some embodiments, the SBPs of the present disclosure may beadministered to treat subjects with diabetic macular edema. In someembodiments, the SBPs of the present disclosure may be used to treatdiabetic retinopathy in subjects with DME. In some embodiments, DME isnon-proliferative. In some embodiments, diabetic retinopathy isnon-proliferative (NPDR). In some embodiments SBPs of the presentdisclosure may be used to achieve the sustained release of one or moreknown NSAID with intravitreal triamcinolone (IVT). In some embodiments,SBPs of the present disclosure may be used to achieve the sustainedrelease of one or more known NSAID with intravitreal triamcinoloneacetonide. In some embodiments, the SBP comprises one or more NSAID andis administered alongside intravitreal triamcinolone or triamcinoloneacetonide. In some embodiments, the SBP comprises one or more NSAID andtriamcinolone or triamcinolone acetonide. In some embodiments, themechanism of action of the treatment is novel compared to that ofexisting treatments of NPDR (e.g. VEGF or steroids). In someembodiments, the mechanism of action of the treatment is additive tothat of VEGF antagonist with respect to the mean improvement in BCVAETDRS. In some embodiments, the mechanism of action of the treatment isadditive to that of VEGF alone with respect to the mean improvement inBCVA ETDRS. In some embodiments, the efficacy of the treatment issimilar to that of intravitreal triamcinolone or triamcinoloneacetonide. In some embodiments, the efficacy of the treatment isimproved over that of intravitreal triamcinolone or triamcinoloneacetonide. In some embodiments, the safety of the treatment is improvedover that of intravitreal triamcinolone or triamcinolone acetonide. Insome embodiments, the adverse event burden is better or similar to thatof a VEGF antagonist. In some embodiments, the adverse event burden isbetter than that of an IVT steroid. In some embodiments, the SBP isadministered via injection. In some embodiments, the SBP is administeredevery 6 months. In some embodiments, the SBP is administered every 3months.

In some embodiments, subjects with NPDR may be evaluated as a part of apopulation of subjects with DME. In some embodiments, SBPs of thepresent disclosure may be administered adjunctive with a VEGFantagonist. In some embodiments, SPBs of the present disclosure may beadministered adjunctive with VEGF and/or VEGF sub-optimal responders. Insome embodiments, treatment of DME and DME in subjects with NPDR may bemeasured by refraction and Best Corrected Visual Acuity using EarlyTreatment in Diabetic Retinopathy Study Methodology (BCVA ETDRS). Insome embodiments, treatment is measured by the mean change in BCVA ETDRSscore at 9 months. In some embodiments, the treatment with SBPs resultsin an improvement in NPDR score. In some embodiments, the improvement isat least two steps.

Methods of treating subjects with ocular SBPs may include one or more oforal administration, intravenous administration, topical administration,and ocular administration. Ocular administration may include one or moreof intravitreal administration, intraretinal administration, intracomealadministration, intrascleral administration, administration to theanterior segment, administration to the posterior segment, andintra-aqueous humor administration. In some embodiments, the SBP adheresto the ocular surface. In some embodiments, the SBP adheres to theocular surface in a manner similar to a mucin layer. Intravitrealadministration may include intravitreal injection. Intravitrealadministration may be performed at any injection site that would enablethe administration of the SBP to the intravitreal space. Intravitrealinjection may be performed by pushing a wire through a syringe andneedle or cannula loaded with ocular SBP. The wire may be pushed untilit extends past the needle or cannula.

In some embodiments, the residence time of an SBP will be analyzed afterSBP administration, using any method known to one skilled in the art. Insome embodiments, the efficacy of an SBP will be analyzed after SBPadministration, using any method known to one skilled in the art. Insome embodiments, the pharmacokinetics of an SBP will be analyzed afterSBP administration, using any method known to one skilled in the art. Insome embodiments, the irritability of an SBP will be analyzed after SBPadministration, using any method known to one skilled in the art. Insome embodiments, the use of an SBP to treat irritation will be analyzedafter SBP administration, using any method known to one skilled in theart. In some embodiments, the toxicity of an SBP will be analyzed afterSBP administration, using any method known to one skilled in the art.

Ocular SBPs may be used to treat subjects by delivering oculartherapeutic agents at a dose of from about 0.01 μg to about 1 μg, fromabout 0.05 μg to about 2 μg, from about 1 μg to about 5 μg, from about 2μg to about 10 μg, from about 4 μg to about 16 μg, from about 5 μg toabout 20 μg, from about 8 μg to about 24 μg, from about 10 μg to about30 μg, from about 12 μg to about 32 μg, from about 14 μg to about 34 μg,from about 16 μg to about 36 μg, from about 18 μg to about 38 μg, fromabout 20 μg to about 40 μg, from about 22 μg to about 42 μg, from about24 μg to about 44 μg, from about 26 μg to about 46 μg, from about 28 μgto about 48 μg, from about 30 μg to about 50 μg, from about 35 μg toabout 55 μg, from about 40 μg to about 60 μg, from about 45 μg to about65 μg, from about 50 μg to about 75 μg, from about 60 μg to about 240μg, from about 70 μg to about 350 μg, from about 80 μg to about 400 μg,from about 90 μg to about 450 μg, from about 100 μg to about 500 μg,from about 200 μg to about 750 μg, from about 300 μg to about 1000 μg,from about 1 μg to about 5000 μg, or from about 500 μg to about 5000 μg.In some embodiments, subjects are contacted with a dose of oculartherapeutic agents sufficient to achieve concentrations in subject eyes(or components of subject eyes) greater than or equal to the effectiveconcentration for such ocular therapeutic agents. The concentrations maybe 1.5-fold, 2-fold, 4-fold, 5-fold, 10-fold, or more than 10-foldgreater than the effective concentration.

In some embodiments, contacting subjects with ocular SBPs results inocular therapeutic agent concentrations in subject eyes of from about0.01 ng/mL to about 70,000 ng/ml. In some embodiments, the resultingconcentration in subject eyes is from about 0.01 ng/mL to about 1 ng/mL,from about 0.05 ng/mL to about 2 ng/mL, from about 1 ng/mL to about 5ng/mL, from about 2 ng/mL to about 10 ng/mL, from about 4 ng/mL to about16 ng/mL, from about 5 ng/mL to about 20 ng/mL, from about 8 ng/mL toabout 24 ng/mL, from about 10 ng/mL to about 30 ng/mL, from about 12ng/mL to about 32 ng/mL, from about 14 ng/mL to about 34 ng/mL, fromabout 16 ng/mL to about 36 ng/mL, from about 18 ng/mL to about 38 ng/mL,from about 20 ng/mL to about 40 ng/mL, from about 22 ng/mL to about 42ng/mL, from about 24 ng/mL to about 44 ng/mL, from about 26 ng/mL toabout 46 ng/mL, from about 28 ng/mL to about 48 ng/mL, from about 30ng/mL to about 50 ng/mL, from about 35 ng/mL to about 55 ng/mL, fromabout 40 ng/mL to about 60 ng/mL, from about 45 ng/mL to about 65 ng/mL,from about 50 ng/mL to about 75 ng/mL, from about 60 ng/mL to about 240ng/mL, from about 70 ng/mL to about 350 ng/mL, from about 80 ng/mL toabout 400 ng/mL, from about 90 ng/mL to about 450 ng/mL, from about 100ng/mL to about 500 ng/mL, from about 0.01 μg/mL to about 1 μg/mL, fromabout 0.05 μg/mL to about 2 μg/mL, from about 1 μg/mL to about 5 μg/mL,from about 2 μg/mL to about 10 μg/mL, from about 4 μg/mL to about 16μg/mL, from about 5 μg/mL to about 20 μg/mL, from about 8 μg/mL to about24 μg/mL, from about 10 μg/mL to about 30 μg/mL, from about 12 μg/mL toabout 32 μg/mL, from about 14 μg/mL to about 34 μg/mL, from about 16μg/mL to about 36 μg/mL, from about 18 μg/mL to about 38 μg/mL, fromabout 20 μg/mL to about 40 μg/mL, from about 22 μg/mL to about 42 μg/mL,from about 24 μg/mL to about 44 μg/mL, from about 26 μg/mL to about 46μg/mL, from about 28 μg/mL to about 48 μg/mL, from about 30 μg/mL toabout 50 μg/mL, from about 35 μg/mL to about 55 μg/mL, from about 40μg/mL to about 60 μg/mL, from about 45 μg/mL to about 65 μg/mL, fromabout 50 μg/mL to about 75 μg/mL, from about 60 μg/mL to about 240μg/mL, from about 70 μg/mL to about 350 μg/mL, from about 80 μg/mL toabout 400 μg/mL, from about 90 μg/mL to about 450 μg/mL, from about 100μg/mL to about 500 μg/mL, from about 0.01 mg/mL to about 1 mg/mL, fromabout 0.05 mg/mL to about 2 mg/mL, from about 1 mg/mL to about 5 mg/mL,from about 2 mg/mL to about 10 mg/mL, from about 4 mg/mL to about 16mg/mL, from about 5 mg/mL to about 20 mg/mL, from about 8 mg/mL to about24 mg/mL, from about 10 mg/mL to about 30 mg/mL, from about 12 mg/mL toabout 32 mg/mL, from about 14 mg/mL to about 34 mg/mL, from about 16mg/mL to about 35 mg/mL, or from about 35 mg/mL to about 70 mg/mL. Theocular therapeutic agent concentration in subject eyes may includeconcentration in one or more eye components. The components may include,but are not limited to, the aqueous humor, vitreous humor, retina,choroid, sclera, lens, fomix, conjunctiva, lacrimal punctum, capsule ofTenon, iris, pupal, cornea, ciliary muscle, fovea, optic nerve, macula,blood vessel, anterior chamber, posterior chamber, and sub-tenon space.In some embodiments, contacting subjects with ocular SBPs may result inocular therapeutic agent concentration in subject aqueous humor of fromabout 0.01 ng/mL to about 2.0 ng/mL. In some embodiments, vitreous humorconcentration may be from about 10 ng/mL to about 20,000 ng/ml. In someembodiments, retina and/or choroid concentrations may be from about 10ng/mL to about 70,000 ng/mL. Ocular therapeutic agent levels may bedetectable in subject eyes for at least 1 day, for at least 2 days, forat least 3 days, for at least 1 week, for at least 2 weeks, for at least1 month, for at least 3 months, for at least 6 months, or for at least 1year. In some embodiments, ocular therapeutic agent levels remain at asteady level for at least 1 day, for at least 2 days, for at least 3days, for at least 1 week, for at least 2 weeks, for at least 1 month,for at least 3 months, for at least 6 months, or for at least 1 year. Insome embodiments, the concentration of the ocular therapeutic agent inthe subject eye or component of the eye is at a level at or near theeffective concentration. In some embodiments, the concentration of theocular therapeutic agent in the subject eye or component of the eye issustained at a level at or near the effective concentration. In someembodiments, the concentration of the ocular therapeutic agent in thesubject eye or component of the eye is sustained at a level greater thanthe effective concentration. In some embodiments the effectiveconcentration is the IC₅₀, the EC₃₀, or the EC₈₀.

In some embodiments, the ocular SBPs may be hydrogels. In someembodiments, the ocular SBPs are rods. In some embodiments, the ocularSBPs are administered via intravitreal administration. In someembodiments, the ocular SBPs are formulated with celecoxib. In someembodiments, the intravitreal administration of the ocular SBPs enablesat least 3 months of sustained release at or above the effectiveconcentration of celecoxib. In some embodiments, the intravitrealadministration of the ocular SBPs enables at least 6 months of sustainedrelease at or above the effective concentration of celecoxib. In someembodiments the effective concentration is the IC₅₀. In someembodiments, the effective concentration is the EC₈₀. In someembodiments, the IC₅₀ is 40 nM. In some embodiments, the EC₈₀ is 1-3 μM.

In some embodiments, ocular SBPs may be used to reduce ocular pressure.In some embodiments, the intravitreal administration of the ocular SBPsresults in a sustained intraocular pressure. In some embodiments, thereduced or sustained intraocular pressure may be observed for at least 1day, at least 3 days, at least 1 week, at least 2 weeks, at least 1month, at least 3 months, at least 4 months, at least 6 months, or atleast 1 year after SBP administration.

In some embodiments, the ocular SBPs of the present disclosure arebiocompatible in the ocular space. In some embodiments, administrationof the ocular SBP does not cause local inflammation in the ocular space.In some embodiments, the SBP is tolerable in the ocular space. In someembodiments, the retinal tissue remains normal after the administrationof the ocular SBP. In some embodiments, the SBPs are biocompatible andtolerable in the ocular space for at least 1 day, at least 3 days, atleast 1 week, at least 2 weeks, at least 1 month, at least 3 months, atleast 4 months, at least 6 months, or at least 1 year.

In some embodiments, the present disclosure provides methods ofdelivering ocular therapeutic agents to subjects by contacting subjecteyes with ocular SBPs. Such ocular SBPs may be prepared by combiningprocessed silk with ocular therapeutic agents. The SBPs may be preparedwith a low temperature, aqueous processing procedure. The SBPs may beprepared as rods. The rods may be prepared by extrusion through a tube.The tube may be a needle. Extrusion may be carried out using a syringe.Ocular therapeutic agents may be delivered to subject eyes by releasefrom SBPs while SBPs are in contact with the eyes. Release of oculartherapeutic agents from SBPs may be modulated by one or more of silkfibroin concentration, silk fibroin molecular weight, SBP volume, methodused to dry SBPs, ocular therapeutic agent molecular weight, andinclusion of at least one excipient. Methods used to dry SBPs mayinclude one or more of oven drying, lyophilizing, and air drying. Insome embodiments, an ocular SBP is prepared as a gel, before drying toobtain the SBP in a rod format. Ocular SBP rods may include oculartherapeutic agents and silk fibroin at a w/w ratio of from about 1 toabout 5.

Release of ocular therapeutic agents from ocular SBPs may occur at arate that includes an initial burst. From about 0.01% to about 100% ofocular therapeutic agents may be released from SBPs during an initialrelease period associated with the initial burst. In some embodiments,from about 5% to about 20% of ocular therapeutic agents may be releasedfrom SBPs during an initial release period associated with the initialburst. Release of ocular therapeutic agent from SBPs may include a dailyrelease percentage of from about 0.1% (w/w) to about 5% (w/w). In someembodiments the release rates of the therapeutic agents are tunable. Insome embodiments, the release rates are tunable on the order of days toweeks. In some embodiments the release rates are tunable on the order ofweeks to months.

In some embodiments, the release rates are tuned by varying the APIloading, the silk fibroin molecular weight, the silk fibroinconcentration, the drying method, and the density of the ocular SBPduring formulation. In some embodiments, the release kinetics of an APIfrom an SBP may be tuned by the density of the SBP. In some embodiments,the daily release percentage and the initial burst may be decreased bypreparation of a denser SBP. In some embodiments, the release kineticsof an API from an SBP may be tuned by the concentration of processedsilk in the SBP. In some embodiments, the daily release percentage andthe initial burst may be decreased by preparation with a higherconcentration of processed silk. In some embodiments, the release of anAPI from an ocular SBP is biphasic, in that the release rate changesbetween two portions of the study.

In some embodiments, from about 1% to about 100% of ocular therapeuticagents are released from ocular SBPs during a release period. Therelease period may be from about 1 day to about 10 months. The releaseperiod may begin upon contacting an eye of a subject with an SBP. Therelease period may be from about 1 day to about 5 months. The releaseperiod may be from about 1 day to about 6 months. In some embodiments,the API is released over a period of at least 1 day, for at least 2days, for at least 3 days, for at least 1 week, for at least 2 weeks,for at least 1 month, for at least 3 months, for at least 6 months, orfor at least 1 year. In some embodiments, 0.1%-100% of oculartherapeutic agents may be released from SBPs over release periods. Insome embodiments, from about 40% to about 60% of ocular therapeuticagents may be released from SBPs over release periods. The oculartherapeutic agents may be released from the ocular SBPs via diffusion,degradation, and/or solvent penetration. In some embodiments, therelease of the therapeutic agents from ocular SBPs follows first orderkinetics. In some embodiments, the release of therapeutic agents fromocular SBPs follows zero order kinetics. In some embodiments the releaseperiods of the therapeutic agents are tunable. In some embodiments, therelease rates are tunable on the order of days to weeks. In someembodiments the release periods are tunable on the order of weeks tomonths. In some embodiments, the release periods are tuned by varyingthe API loading, API hydrophobicity. API molecular weight, the silkfibroin molecular weight, the silk fibroin concentration, thehydrophobicity of the SBP formulation, depot surface area, matrixporosity/density, and/or the density of the ocular SBP duringformulation. In some embodiments, the therapeutic agent is an NSAID. Insome embodiments, the SBP formulated with NSAID has a release period ofat least 1 day, at least 3 days, at least 1 week, at least 1 month, atleast 3 months, at least 6 months, or at least 1 year in vitro. In someembodiments, the SBP formulated with NSAID has a release period of atleast 1 day, at least 3 days, at least 1 week, at least 1 month, atleast 3 months, at least 6 months, or at least 1 year in vivo.

In some embodiments, the ocular SBP is a rod, and the release durationof CXB is related to the rod density. In some embodiments, increaseddensity of a rod results in increased release times. In someembodiments, the density of the rod is tuned by varying the startingconcentration of the silk-fibroin used during formulation. In someembodiments, the rods with a density below 1.0 g/mL reach completerelease about 64 days or less. In some embodiments, the rods with adensity between 1.0 g/mL and 1.1 g/mL reach complete release in about 98days. In some embodiments, the rods with a density above 1.1 g/mL reachcomplete release in greater than 98 days.

In some embodiments, the ocular SBP may comprise one of more of thefollowing components: silk fibroin, one or more excipients, anonsteroidal anti-inflammatory drug, and Tween-80. Such ocular SBP maybe formatted as hydrogels or rods.

In some embodiments, the ocular SBP comprises one of more of thefollowing components: silk fibroin, poloxamer 188 (P188), celecoxib(CXB), phosphate buffer, and Tween-80. The silk fibroin may be presentin a concentration between 1 to 5% (w/v). The silk fibroin may have aminute boil in the range of 120 to 480. Poloxamer 188 (P188) may bepresent in a concentration between 1 to 30% (w/v). Celecoxib (CXB) maybe present in a concentration between 0.1 to 20% (w/v). The phosphatebuffer may be present in the concentration between 10 to 30 mM. Thephosphate buffer may have a pH between 7.2 to 7.6. Tween-80 may bepresent in a concentration between 0.1 to 0.5%. The ocular SBP may havean osmolarity between 280 to 300 mOsm/L. Such ocular SBP may beformatted as hydrogels.

As a non-limiting example, the ocular SBP comprises about 3% (w/v) silkfibroin with a 480-minute boil, about 10% poloxamer 188 (P188), about10% celecoxib (CXB), about 22 mM phosphate buffer with a pH of 7.4, andabout 0.2% Tween-80, and the ocular SBP has an osmolarity of about 290mOsm/L and is formatted as hydrogels.

II. Therapeutic Applications

In some embodiments, SBPs may be used in a variety of therapeuticapplications. As used herein, the term “therapeutic application” refersto any method related to restoring or promoting the health, nutrition,and/or wellbeing of a subject; supporting or promoting reproduction in asubject; or treating, preventing, mitigating, alleviating, curing, ordiagnosing a disease, disorder, or condition. As used herein, the term“condition” refers to a physical state of wellbeing. Therapeuticapplications may include, but are not limited to, medical applications,surgical applications, and veterinary applications. As used herein, theterm “medical application” refers to any method or use that involvestreating, diagnosing, and/or preventing disease according to the scienceof medicine. “Surgical applications” refer to methods of treatmentand/or diagnosis that involve operation on a subject, typicallyrequiring incision and the use of instruments. “Veterinary applications”refer to therapeutic applications where the subject is a non-humananimal. In some embodiments, therapeutic applications may include, butare not limited to, experimental, diagnostic, or prophylacticapplications. In some embodiments, therapeutic applications includepreparation and/or use of therapeutic devices. As used herein, the term“therapeutic device” refers to any article prepared or modified fortherapeutic use.

SBPs used for therapeutic applications may include or may be combinedwith one or more pharmaceutical compositions, implants, therapeuticagents, coatings, excipients, or devices. In some embodiments, SBPsfacilitate the delivery and/or controlled release of therapeutic agentpayloads. In some embodiments, SBPs described herein may be used tostabilize therapeutic agents. Some SBPs may be used as tools, materials,or devices in therapeutic applications. Such SBPs may include, but arenot limited to, delivery vehicles, and scaffolds.

Subjects

Therapeutic applications of the present disclosure may be applied to avariety of subjects. As used herein, the term “subject” refers to anyentity to which a particular process or activity relates to or isapplied. Subjects of therapeutic applications described herein may behuman or non-human. Human subjects may include humans of different ages,genders, races, nationalities, or health status. Non-human subjects mayinclude non-human animal subjects (also simply referred to herein as“animal subjects”). Animal subjects may be non-human vertebrates orinvertebrates. Some animal subjects may be wild type or geneticallymodified organisms (e.g., transgenic). In some embodiments, subjectsinclude patients. As used herein, the term “patient” refers to a subjectseeking treatment, in need of treatment, requiring treatment, receivingtreatment, expecting treatment, or who is under the care of a trained(e.g., licensed) professional for a particular disease, disorder, and/orcondition.

Veterinary Applications

In some embodiments, SBPs may be used in veterinary applications torestore or promote the health and/or wellbeing of a non-human animalsubject and/or to treat, prevent, alleviate, cure, or diagnose adisease, disorder, or condition of a non-human animal subject. As anon-limiting example, the SBPs may be used for companion animal health.As another non-limiting example, the SBPs may be used for farm animalhealth.

In one embodiment, the veterinary indication is dry eye disease.

Therapeutic Agents

In some embodiments, therapeutic applications involve the use of SBPsthat are therapeutic agents or are combined with one or more therapeuticagents. As used herein, the term “therapeutic agent” refers to anysubstance used to restore or promote the health and/or wellbeing of asubject and/or to treat, prevent, alleviate, cure, or diagnose adisease, disorder, or condition. Examples of therapeutic agents include,but are not limited to, adjuvants, analgesic agents, antiallergicagents, antiangiogenic agents, antiarrhythmic agents, antibacterialagents, antibiotics, antibodies, anticancer agents, anticoagulants,antidementia agents, antidepressants, antidiabetic agents, antigens,antihypertensive agents, anti-infective agents, anti-inflammatoryagents, antioxidants, antipyretic agents, anti-rejection agents,antiseptic agents, antitumor agents, antiulcer agents, antiviral agents,biological agents, birth control medication, carbohydrates,cardiotonics, cells, chemotherapeutic agents, cholesterol loweringagents, cytokines, endostatins, enzymes, fats, fatty acids, geneticallyengineered proteins, glycoproteins, growth factors, health supplements,hematopoietics, herbal preparations, hormones, hypotensive diuretics,immunological agents, inorganic synthetic pharmaceutical drugs, ions,lipoproteins, metals, minerals, nanoparticles, naturally derivedproteins, NSAIDs, nucleic acids, nucleotides, organic syntheticpharmaceutical drugs, oxidants, peptides, pills, polysaccharides,proteins, protein-small molecule conjugates or complexes, psychotropicagents, small molecules, sodium channel blockers, statins, steroids,stimulants, therapeutic agents for osteoporosis, therapeuticcombinations, thrombopoietics, tranquilizers, vaccines, vasodilators,VEGF-related agents, veterinary agents, viruses, virus particles, andvitamins. Other therapeutic agents may include, but are not limited to,anthocyanidin, anthoxanthin, apigenin, dihydrokaempferol, eriodictyol,fisetin, flavan, flavan-3,4-diol, flavan-3-ol, flavan-4-ol, flavanone,flavanonol, flavonoid, furanoflavonols, galangin, hesperetin,homoeriodictyol, isoflavonoid, isorhamnetin, kaempferol, luteolin,myricetin, naringenin, neoflavonoid, pachypodol, proanthocyanidins,pyranoflavonols, quercetin, rhamnazin, tangeritin, taxifolin,theaflavin, thearubigin, chondrocyte-derived extracellular matrix,macrolide, erythromycin, roxithromycin, azithromycin and clarithromycin.In some embodiments, SBP therapeutics and methods of delivery mayinclude any of those taught in International Publication NumbersWO2017139684, WO2010123945, WO2017123383, or United States PublicationNumbers US20170340575, US20170368236, and US20110171239 the contents ofeach of which are herein incorporated by reference in their entirety.

Processed Silk as a Therapeutic Agent

In some embodiments, SBPs that consist of or include processed silk areused as therapeutic agents, wherein processed silk is an activetherapeutic component. The processed silk may include, but is notlimited to one or more of silk fibroin, fragments of silk fibroin,chemically altered silk fibroin, and mutant silk fibroin. Therapeuticapplications including such SBPs may include any of those taught inInternational Publication Number WO2017200659; Aykac et al. (2017) Genes0378-1119(17)30865-8; and Abdel-Naby (2017) PLoS One 12(11):e0188154,the contents of each of which are herein incorporated by reference intheir entirety. Processed silk may be administered as a therapeuticagent for treatment of a localized indication or for treatment of anindication further from the SBP application site. In some embodiments,therapeutic agents are combinations of processed silk and some otheractive component. In some embodiments, therapeutic agent activityrequires cleavage or dissociation from silk. Therapeutic agents mayinclude silk fibroin and/or chemically modified silk fibroin. In someembodiments, such therapeutic agents may be used to treat burn injury,inflammation, wound healing, or corneal injury. These and othertreatments may be carried out according to any of the methods describedin International Publication Number WO2017200659; United StatesPublication Number US20140235554; Aykac et al. (2017) Genes0378-1119(17)30868-30865; or Abdel-Naby (2017) PLoS One12(11):e0188154, the contents of each of which are herein incorporatedby reference in their entirety. In some embodiments, SBPs are silkfibroin solutions used to facilitate wound healing, as described in Parket al. (2017) Acta Biomater 67:183-195, the contents of which are hereinincorporated by reference in their entirety. These SBPs may enhancewound healing via a nuclear factor kappa enhancer binding protein(NF-κB) signaling pathway. In some embodiments, SBPs are therapeuticagents used to facilitate delivery and/or release of therapeutic agentpayloads. Such therapeutic agents and/or methods of use may include, butare not limited to, any of those described in International PublicationNumber WO2017139684, the contents of which are herein incorporated byreference in their entirety.

Lubricants

In some embodiments, processed silk and/or SBPs may be used as alubricant. In some embodiments, processed silk may be selected base onor prepared to maximize its use as a lubricant. As used herein, the term“lubricant” refers to a substance that reduces the friction between twoor more surfaces. In some embodiments, the surfaces in need oflubrication may be part of a subject. In some embodiments, surfaces inneed of lubrication include, but are not limited to, the body, eyes,skin, scalp, mouth, vagina, nose, hands, feet, and lips. In someembodiments, SBPs are used for ocular lubrication. As used herein, theterm “ocular lubrication” refers to a method of the reduction offriction and/or irritation in the eye. In some embodiments, processedsilk and/or SBPs may be used to reduce friction caused by dryness, astaught in U.S. Pat. No. 9,907,836 (the content of which is hereinincorporated by reference in its entirety). This dryness may be drynessin the eye.

In some embodiments, the coefficient of friction of an SBP isapproximately that of naturally occurring, biological and/or proteinlubricants (e.g. lubricin). In some embodiments. SBPs may beincorporated into a lubricant. Such methods may include any of thosepresented in International Publication No. WO2013163407, the contents ofwhich are herein incorporated by reference in their entirety. In someembodiments, processed silk and/or SBPs may be used as an excipient. Insome embodiments, processed silk and/or SBPs may be used as an excipientto prepare a lubricant.

Biological Agents

In some embodiments, therapeutic agents include biological agents (alsoreferred to as “biologics” or “biologicals”). As used herein, a“biological agent” refers to a therapeutic substance that is or isderived from an organism or virus. Examples of biological agentsinclude, but are not limited to, proteins, organic polymers andmacromolecules, carbohydrates, complex carbohydrates, nucleic acids,cells, tissues, organs, organisms, DNA, RNA, oligonucleotides, genes,and lipids. Biological agents may include processed silk.

In some embodiments, SBPs may be used to deliver or administerbiological agents. In some embodiments, delivery may include controlledrelease of one or more biological agents. Delivery may be carried out invivo. In some embodiments, delivery is in vitro. Processed silk may beused to facilitate delivery and/or maintain stability of biologicalagents.

In some embodiments, SBPs are used to deliver proteins. Non-limitingexamples of proteins that may be delivered with SBPs include monoclonalantibodies, immunoglobulins (e.g., IgG), anti-VEGF antibodies (e.g.,AVASTIN®), lysozyme, and bovine serum albumin (BSA). SBPs may providecontrolled release of a stable protein over a desired administrationperiod, for example, for at least 1 day, at least 2 days, at least 3days, at least 4 days, at least 5 days, at least 6 days, at least 7days, at least 8 days, at least 9 days, at least 10 days, at least 11days, at least 12 days, at least 13 days, at least 2 weeks, at least 3weeks, at least 1 month, at least 6 weeks, at least 2 months, at least10 weeks, at least 3 months, at least 6 months, at least 9 months, or atleast 1 year. In one embodiment, SBPs provide controlled release of astable protein for at least 7 days.

SBP formulations used for protein delivery may be tailored based onvariables such as the molecular weight of the protein to be delivered,the loading of the protein, the molecular weight of the silk fibroin,and the silk fibroin concentration used in the formulations.

Ocular Therapeutic Agents

In some embodiments, therapeutic agents include ocular therapeuticagents. As used herein, the term “ocular therapeutic agent” refers toany compound that has a healing, corrective, diagnostic, and/orprophylactic effect and/or elicits a desired biological and/orpharmacological effect on the eye. In some embodiments, oculartherapeutic agents include one or more of processed silk, biologicalagents, small molecules, proteins, nonsteroidal anti-inflammatory drugs,and vascular endothelial growth factor-related agents. Oculartherapeutic agent proteins may include, but are not limited to,lysozyme, bovine serum albumin (BSA), bevacizumab, or VEGF-relatedagents. In some embodiments, ocular therapeutic agents may be used totreat one or more of the ocular therapeutic indications describedherein.

Nonsteroidal Anti-Inflammatory Drugs

Therapeutic agents may include nonsteroidal anti-inflammatory drugs. Anonsteroidal anti-inflammatory drug (NSAID) is a class of non-opioidanalgesics used to reduce inflammation and associated pain. NSAIDs mayinclude small molecules. NSAIDs may include, but are not limited to,aspirin, carprofen, celecoxib, deracoxib, diclofenac, diflunisal,etodolac, fenoprofen, firocoxib, flurbirofen, ibuprofen, indomethacin,ketoprofen, ketorolac, mefenamic acid, meloxicam, nabumetone, naproxen,oxaprozin, piroxicam, robenacoxib, salsalate, sulindac, and tolmetin. Insome embodiments, NSAIDs may be used to treat one or more of the oculartherapeutic indications described herein. In some embodiments, the NSAIDis celecoxib. Some SBPs include gels or hydrogels that are combined withNSAIDs (e.g., celecoxib). Such SBPs may be used as carriers for NSAIDpayload delivery. NSAID delivery may include controlled release of theNSAID.

Vascular Endothelial Growth Factor-Related Agents

In some embodiments, therapeutic agents include modulators ofangiogenesis. Such therapeutic agents may include vascular endothelialgrowth factor (VEGF)-related agents. As used herein, the term“VEGF-related agent” refers to any substance that affects VEGFexpression, synthesis, stability, biological activity, degradation,receptor binding, cellular signaling, transport, secretion,internalization, concentration, or deposition (e.g., in extracellularmatrix). In some embodiments, VEGF-related agents may be used to treatone or more of the ocular therapeutic indications described herein.

In some embodiments, VEGF-related agents are angiogenesis inhibitors. Insome embodiments, the angiogenesis inhibitor includes any of thosetaught in International Publication Number WO2013126799, the contents ofwhich are herein incorporated by reference in their entirety. In someembodiments, VEGF-related agents may include antibodies. VEGF-relatedagents may include VEGF agonists, including, but not limited to,toll-like receptor agonists. In some embodiments, the therapeutic agentis a VEGF antagonist. VEGF agonists or antagonists may be smallmolecules. In some embodiments, VEGF agonists or antagonists may bemacromolecules or proteins. Angiogenesis inhibitors may include, but arenot limited to. MACUGEN® or another VEGF nucleic acid ligand; LUCENTIS®,AVASTIN®, or another anti-VEGF antibody; combretastatin or a derivativeor prodrug thereof such as Combretastatin A4 Prodrug (CA4P); VEGF-Trap(Regeneron); EVIZON™ (squalamine lactate); AG-013958 (Pfizer, Inc.);JSM6427 (Jerini AG); a short interfering RNA (siRNA) that inhibitsexpression of one or more VEGF isoforms (e.g., VEGF₁₆₅); an siRNA thatinhibits expression of a VEGF receptor (e.g., VEGFRl), endogenous orsynthetic peptides, angiostatin, combstatin, arresten, tumstatin,thalidomide, thalidomide derivatives, canstatin, endostatin,thrombospondin, and β2-glycoprotein 1.

Therapeutic Indications

In some embodiments, SBPs may be used to address one or more therapeuticindications. As used herein, the term “therapeutic indication” refers toa disease, disorder, condition, or symptom that may be cured, reversed,alleviated, stabilized, improved, or otherwise addressed through someform of therapeutic intervention (e.g., administration of a therapeuticagent or method of treatment).

SBP treatment of therapeutic indications may include contacting subjectswith SBPs. SBPs may include therapeutic agents (e.g., any of thosedescribed herein) as cargo or payloads for treatment. In someembodiments, payload release may occur over a period of time (the“payload release period”). The payload release rate and/or length of thepayload release period may be modulated by SBP components or methods ofpreparation.

Ocular Indications

In some embodiments, therapeutic indications include ocular indications.As used herein, the term “ocular indication” refers to any therapeuticindication related to the eye. In some embodiments, the therapeuticindication is an ophthalmology or ophthalmology-related disease and/ordisorder. Treatment of such indications in subjects may includecontacting subjects with SBPs. SBPs may include therapeutic agents(e.g., any of those described herein) as cargo or payloads fortreatment. In some embodiments, payload release may occur over a periodof time (the payload release period). The payload release rate and/orlength of the payload release period may be modulated by SBP componentsor methods of preparation. In some embodiments, SBPs may be provided inthe form of a solution or may be incorporated into a solution for ocularadministration. Such solutions may be administered topically (e.g., inthe form of drops, creams, or sprays) or by injection. In someembodiments, SBPs may be provided in the format of a lens or may beincorporated into lenses that are placed on eye. In some embodiments,SBPs are provided in the form of implants or are incorporated intoimplants that may be placed around the eye, on a surface of the eye, ina periocular space or compartment, or intraocularly. Implants may besolid or gelatinous (e.g., a gel or slurry) and may be in the form of ableb, rod, or plug. Some gelatinous implants may harden afterapplication. In some embodiments, implants include punctal plugs. Suchplugs may be inserted into tear ducts. In some embodiments, SBPs may beused to repair ocular damage. In some embodiments, the SBP adheres tothe ocular surface. In some embodiments, the SBP adheres to the ocularsurface in a manner similar to a mucin layer.

Non-limiting examples of ocular indications include infection,refractive errors, age related macular degeneration, cystoid macularedema, cataracts, diabetic retinopathy (proliferative andnon-proliferative), glaucoma, amblyopia, strabismus, color blindness,cytomegalovirus retinitis, keratoconus, diabetic macular edema(proliferative and non-proliferative), low vision, ocular hypertension,retinal detachment, eyelid twitching, inflammation, uveitis, bulgingeyes, dry eye disease, floaters, xerophthalmia, diplopia, Graves'disease, night blindness, eye strain, red eyes, nystagmus, presbyopia,excess tearing, retinal disorders (e.g. age related maculardegeneration), conjunctivitis, cancer, comeal ulcer, comeal abrasion,snow blindness, scleritis, keratitis, Thygeson's superficial punctatekeratopathy, corneal neovascularization, Fuch's dystrophy,keratoconjuctitivis sicca, iritis, chorioretinal inflammation (e.g.chorioretinitis, choroiditis, retinitis, retinochoroiditis, parsplanitis, and Harada's disease), aniridia, macular scars, solarretinopathy, choroidal degeneration, choroidal dystrophy, choroideremia,gyrate atrophy, choroidal hemorrhage, choroidal detachment,retinoschisis, hypertensive retinopathy. Bull's eye maculopathy,epiretinal membrane, peripheral retinal degeneration, hereditary retinaldystrophy, retinitis pigmentosa, retinal hemorrhage, separation ofretinal layers, retinal vein occlusion, and other visual impairments. Insome embodiments, ocular indications include inflammation of the eye.

Ocular indications may include dry eye. Dry eye is a condition involvinga lack of hydration on the eye surface that may be caused by one or moreof a variety of factors (e.g., cellular/tissue dysfunction orenvironmental irritants). In some embodiments, SBPs used to treat dryeye are provided as or included in solutions or devices. Solutions maybe administered topically (e.g., by cream, spray, or drops) or byinjection to periocular or intraocular areas. Solutions may includeviscous solutions, such as gels or slurries. Devices may include, butare not limited to, implants, lenses, and plugs. Devices may be hardenedstructures or gelatinous. In some embodiments, devices are gelatinous,but harden after placement. Devices may include lacrimal or punctalplugs that treat dry eye via tear duct insertion. SBPs used to treat dryeye may include therapeutic agent payloads. The therapeutic agents mayinclude any of those described herein. In some embodiments, therapeuticagents include one or more of cyclosporine, corticosteroids,tetracyclines, and essential fatty acids. Therapeutic agent release fromSBPs may occur over an extended payload release period. The payloadrelease period may be from about 1 hour to about 48 hours, from about 1day to about 14 days, or from about 1 week to about 52 weeks, or morethan 52 weeks. In some embodiments, ocular SBPs may be used as ananti-inflammatory treatment for dry eye disease, as described in Kim etal. (2017) Scientific Reports 7: 44364, the contents of which are hereinincorporated by reference in their entirety. It has been demonstratedthat the administration of 0.1 to 0.5% silk fibroin solutions in a mousemodel of dry eye disease enhances comeal smoothness and tear production,while reducing the amount of inflammatory markers detected.

Ocular indications may include glaucoma. The term “glaucoma” refers to agroup of ocular disorders that cause optic nerve damage, sometimesleading to loss of vision or blindness. Glaucoma is often associatedwith elevated intraocular pressure. The pressure may be caused byinefficient drainage of intraocular fluid. The optic nerve is sensitiveto intraocular pressure and increased pressure can lead to damage.“Refractory glaucoma” refers to glaucoma that persists or is at risk topersist after attempts to reduce intraocular pressure (e.g., surgicalintervention).

Ocular indications may include diabetic retinopathy. The term “diabeticretinopathy” refers to the damage to the blood vessels in the back ofthe eye caused by complications of diabetes. Both type I and type IIdiabetes can lead to diabetic retinopathy. The early stages of theindication, known as non-proliferative diabetic retinopathy, includeweakened blood vessels and microaneurysms. The later stages of theindication, known as proliferative diabetic retinopathy, may lead to alack of circulation in the retina and improper blood vessel growth.

Ocular indications may include diabetic macular edema. The term“diabetic macular edema” refers to an accumulation of the fluid in themacula, the area of the eye responsible for high-resolution centralvision. Diabetic macular edema may be caused by diabetic retinopathy.Treatments for diabetic macular edema may include VEGF-related agents(e.g. antibodies or antagonists), and steroids (e.g. triameinolone).

In some embodiments, ocular indications may include cystoid macularedema (CME). CME is caused by cyst-like (cystoid) areas of accumulatedfluid inside the retina in the macular area. Cystoid macular edema canbe diagnosed via dilated retinal exam, fluorescein angiography, oroptical coherence tomography. Current treatment options for CME includelaser therapy, topical nonsteroidal anti-inflammatory drugs (NSAIDs)(e.g., Ketorolac, OCUFEN®, Diclofenac Sodium, PROLENSA®, ILEVRO®, andNEVANAC®), corticosteroids (e.g., triamcinolone), and other therapeuticoptions such as carbonic anhydrase inhibitors, intravitreal anti-VEGFagents, and possibly surgery. SBPs described herein may be used alone orin combination with an existing treatment method for treating CME.

In some embodiments, ocular indications may include post-operativecystoid macular edema (CME). In some embodiments, ocular indications mayinclude age-related macular degeneration (AMD), whether wet or dry. Insome embodiments, ocular indications may include diabetic macular edema(DME).

Combinations

In some embodiments, SBPs may be administered in combination with othertherapeutic agent and/or methods of treatment, e.g., with knownpharmaceuticals and/or known therapeutic methods, such as, for example,those which are currently employed for treating these disorders. Forexample, SBPs used to treat ocular indications may be administered incombination with other therapeutic agents used to treat ocularindications.

Pharmaceutical Compositions

In some embodiments, SBPs are or are included in pharmaceuticalcompositions. As used herein, the term “pharmaceutical composition”refers to a composition designed and/or used for medicinal purposes(e.g., the treatment of a disease and/or disorder).

In some embodiments, pharmaceutical compositions include one or moreexcipients and/or one or more therapeutic agents. Excipients and/ortherapeutic agents included in pharmaceutical compositions may include,but are not limited to, any of those described herein. Relative amountsof therapeutic agents, excipient, and/or any additional ingredients inpharmaceutical compositions may vary, depending upon the identity, size,and/or condition of subjects being treated and further depending uponroutes by which compositions are administered. For example, thecompositions may include from about 0.1% to about 99% (w/w) of atherapeutic agent.

Some excipients may include pharmaceutically acceptable excipients. Thephrase “pharmaceutically acceptable” as used herein, refers tosuitability within the scope of sound medical judgment for contactingsubject (e.g., human or animal) tissues and/or bodily fluids withtoxicity, irritation, allergic response, or other complication levelsyielding reasonable benefit/risk ratios. As used herein, the term“pharmaceutically acceptable excipient” refers to any ingredient, otherthan active agents, that is substantially nontoxic and non-inflammatoryin a subject. Pharmaceutically acceptable excipients may include, butare not limited to, solvents, dispersion media, diluents, inertdiluents, buffering agents, lubricating agents, oils, liquid vehicles,dispersion or suspension aids, surface active agents, isotonic agents,thickening or emulsifying agents, preservatives, and the like, as suitedto the particular dosage form desired. Various excipients forformulating pharmaceutical compositions and techniques for preparing thecomposition are known in the art (see Remington: The Science andPractice of Pharmacy, 21^(st) Edition, A. R. Gennaro, Lippincott,Williams & Wilkins, Baltimore, Md., 2006; incorporated herein byreference in its entirety). The use of a conventional excipient mediummay be contemplated within the scope of the present disclosure, exceptinsofar as any conventional excipient medium may be incompatible with asubstance or its derivatives, such as by producing any undesirablebiological effect or otherwise interacting in a deleterious manner withany other component(s) of pharmaceutical compositions.

A pharmaceutical composition in accordance with the present disclosuremay be prepared, packaged, and/or sold in bulk, as a single unit dose,and/or as a plurality of single unit doses. As used herein, a “unitdose” refers to a discrete amount of the pharmaceutical compositioncomprising a predetermined amount of therapeutic agent or othercompound. The amount of therapeutic agent may generally be equal to thedosage of therapeutic agent administered to a subject and/or aconvenient fraction of such dosage including, but not limited to,one-half or one-third of such a dosage.

In some embodiments, pharmaceutical compositions may include between 20to 55% (w/w) silk fibroin. In some embodiments, the formulations of silkfibroin rods described herein may include between 40 to 80% (w/w)therapeutic agent. In some embodiments, pharmaceutical compositions mayinclude about 33% (w/w) silk fibroin and about 67% (w/w) therapeuticagent. In some embodiments, pharmaceutical compositions may includeabout 25% (w/w) silk fibroin and about 75% (w/w) therapeutic agent. Insome embodiments, pharmaceutical compositions may include about 20%(w/w) silk fibroin and about 80% (w/w) therapeutic agent. In someembodiments, pharmaceutical compositions may include about 40% (w/w)silk fibroin and about 60% (w/w) therapeutic agent. In some embodiments,pharmaceutical compositions may include about 29% (w/w) silk fibroin andabout 71% (w/w) therapeutic agent. In some embodiments, pharmaceuticalcompositions may include about 40% (w/w) silk fibroin and about 60%(w/w) therapeutic agent.

In some embodiments, pharmaceutical compositions may include 35% (w/w)silk fibroin and 65% (w/w) therapeutic agent. In some embodiments,pharmaceutical compositions may include 30% (w/w) silk fibroin and 70%(w/w) therapeutic agent. In some embodiments, pharmaceuticalcompositions may include 40% (w/w) silk fibroin and 60% (w/w)therapeutic agent. In some embodiments, pharmaceutical compositions mayinclude 26% (w/w) silk fibroin and 74% (w/w) therapeutic agent. In someembodiments, pharmaceutical compositions may include 37% (w/w) silkfibroin and 63% (w/) therapeutic agent. In some embodiments,pharmaceutical compositions may include 33% (w/w) silk fibroin and 66%(w/w) therapeutic agent. In some embodiments, pharmaceuticalcompositions may include 51% (w/w) silk fibroin and 49% (w/w)therapeutic agent.

Dosing

In some embodiments, the present disclosure provides methods ofadministering pharmaceutical compositions that are or include SBPs tosubjects in need thereof. Such methods may include providingpharmaceutical compositions at one or more doses and/or according to aspecific schedule. In some embodiments, doses may be determined based ondesired amounts of therapeutic agent or SBP to be delivered. Doses maybe adjusted to accommodate any route of administration effective for aparticular therapeutic application. The exact amount required will varyfrom subject to subject, depending on the species, age, and generalcondition of the subject, the severity of the disease, the particularcomposition, its mode of administration, its mode of activity, and thelike. The frequency of dosing required will also vary from subject tosubject, depending on the species, age, and general condition of thesubject, the severity of the disease, the particular composition, itsmode of administration, its mode of activity, and the like.

SBPs may be formulated in dosage unit form. Such forms may allow forease of administration and uniformity of dosage. Total daily SBP usagemay be decided by an attending physician within the scope of soundmedical judgment. The specific therapeutically effective,prophylactically effective, or appropriate imaging dose level for anyparticular patient will depend upon a variety of factors including thedisorder being treated and the severity of the disorder; the activity ofthe specific compound employed; the specific composition employed: theage, body weight, general health, sex and diet of the patient; the timeof administration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed; andlike factors well known in the medical arts.

In some embodiments, pharmaceutical compositions that are or includeSBPs may include a therapeutic agent or SBP at a concentration of fromabout 10 ng/mL to about 30 ng/mL, from about 12 ng/mL to about 32 ng/mL,from about 14 ng/mL to about 34 ng/mL, from about 16 ng/mL to about 36ng/mL, from about 18 ng/mL to about 38 ng/mL, from about 20 ng/mL toabout 40 ng/mL, from about 22 ng/mL to about 42 ng/mL, from about 24ng/mL to about 44 ng/mL, from about 26 ng/mL to about 46 ng/mL, fromabout 28 ng/mL to about 48 ng/mL, from about 30 ng/mL to about 50 ng/mL,from about 35 ng/mL to about 55 ng/mL, from about 40 ng/mL to about 60ng/mL, from about 45 ng/mL to about 65 ng/mL, from about 50 ng/mL toabout 75 ng/mL, from about 60 ng/mL to about 240 ng/mL, from about 70ng/mL to about 350 ng/mL, from about 80 ng/mL to about 400 ng/mL, fromabout 90 ng/mL to about 450 ng/mL, from about 100 ng/mL to about 500ng/mL, from about 0.01 μg/mL to about 1 μg/mL, from about 0.05 μg/mL toabout 2 μg/mL, from about 1 μg/mL to about 5 μg/mL, from about 2 μg/mLto about 10 μg/mL, from about 4 μg/mL to about 16 μg/mL, from about 5μg/mL to about 20 μg/mL, from about 8 μg/mL to about 24 μg/mL, fromabout 10 μg/mL to about 30 μg/mL, from about 12 μg/mL to about 32 μg/mL,from about 14 μg/mL to about 34 μg/mL, from about 16 μg/mL to about 36μg/mL, from about 18 μg/mL to about 38 μg/mL, from about 20 μg/mL toabout 40 μg/mL, from about 22 μg/mL to about 42 μg/mL, from about 24μg/mL to about 44 μg/mL, from about 26 μg/mL to about 46 μg/mL, fromabout 28 μg/mL to about 48 μg/mL, from about 30 μg/mL to about 50 μg/mL,from about 35 μg/mL to about 55 μg/mL, from about 40 μg/mL to about 60μg/mL, from about 45 μg/mL to about 65 μg/mL, from about 50 μg/mL toabout 75 μg/mL, from about 60 μg/mL to about 240 μg/mL, from about 70μg/mL to about 350 μg/mL, from about 80 μg/mL to about 400 μg/mL, fromabout 90 μg/mL to about 450 μg/mL, from about 100 μg/mL to about 500μg/mL, from about 0.01 mg/mL to about 1 mg/mL, from about 0.05 mg/mL toabout 2 mg/mL, from about 1 mg/mL to about 5 mg/mL, from about 2 mg/mLto about 10 mg/mL, from about 4 mg/mL to about 16 mg/mL, from about 5mg/mL to about 20 mg/mL, from about 8 mg/mL to about 24 mg/mL, fromabout 10 mg/mL to about 30 mg/mL, from about 12 mg/mL to about 32 mg/mL,from about 14 mg/mL to about 34 mg/mL, from about 16 mg/mL to about 36mg/mL, from about 18 mg/mL to about 38 mg/mL, from about 20 mg/mL toabout 40 mg/mL, from about 22 mg/mL to about 42 mg/mL, from about 24mg/mL to about 44 mg/mL, from about 26 mg/mL to about 46 mg/mL, fromabout 28 mg/mL to about 48 mg/mL, from about 30 mg/mL to about 50 mg/mL,from about 40 mg/mL to about 100 mg/mL, or more than 100 mg/mL.

In some embodiments, pharmaceutical compositions that are or includeSBPs may be administered at a dose that provides subjects with a mass oftherapeutic agent or SBP per unit mass of the subject (e.g., mgtherapeutic agent or SBP per kg of subject [mg/kg]). In someembodiments, therapeutic agents or SBPs are administered at a dose offrom about 1 ng/kg to about 5 ng/kg, from about 2 ng/kg to about 10ng/kg, from about 4 ng/kg to about 16 ng/kg, from about 5 ng/kg to about20 ng/kg, from about 8 ng/kg to about 24 ng/kg, from about 10 ng/kg toabout 30 ng/kg, from about 12 ng/kg to about 32 ng/kg, from about 14ng/kg to about 34 ng/kg, from about 16 ng/kg to about 36 ng/kg, fromabout 18 ng/kg to about 38 ng/kg, from about 20 ng/kg to about 40 ng/kg,from about 22 ng/kg to about 42 ng/kg, from about 24 ng/kg to about 44ng/kg, from about 26 ng/kg to about 46 ng/kg, from about 28 ng/kg toabout 48 ng/kg, from about 30 ng/kg to about 50 ng/kg, from about 35ng/kg to about 55 ng/kg, from about 40 ng/kg to about 60 ng/kg, fromabout 45 ng/kg to about 65 ng/kg, from about 50 ng/kg to about 75 ng/kg,from about 60 ng/kg to about 240 ng/kg, from about 70 ng/kg to about 350ng/kg, from about 80 ng/kg to about 400 ng/kg, from about 90 ng/kg toabout 450 ng/kg, from about 100 ng/kg to about 500 ng/kg, from about0.01 μg/kg to about 1 μg/kg, from about 0.05 μg/kg to about 2 μg/kg,from about 1 μg/kg to about 5 μg/kg, from about 2 μg/kg to about 10μg/kg, from about 4 μg/kg to about 16 μg/kg, from about 5 μg/kg to about20 μg/kg, from about 8 μg/kg to about 24 μg/kg, from about 10 μg/kg toabout 30 μg/kg, from about 12 μg/kg to about 32 μg/kg, from about 14μg/kg to about 34 μg/kg, from about 16 μg/kg to about 36 μg/kg, fromabout 18 μg/kg to about 38 μg/kg, from about 20 μg/kg to about 40 μg/kg,from about 22 μg/kg to about 42 μg/kg, from about 24 μg/kg to about 44μg/kg, from about 26 μg/kg to about 46 μg/kg, from about 28 μg/kg toabout 48 μg/kg, from about 30 μg/kg to about 50 μg/kg, from about 35μg/kg to about 55 μg/kg, from about 40 μg/kg to about 60 μg/kg, fromabout 45 μg/kg to about 65 μg/kg, from about 50 μg/kg to about 75 μg/kg,from about 60 μg/kg to about 240 μg/kg, from about 70 μg/kg to about 350μg/kg, from about 80 μg/kg to about 400 μg/kg, from about 90 μg/kg toabout 450 μg/kg, from about 100 μg/kg to about 500 μg/kg, from about0.01 mg/kg to about 1 mg/kg, from about 0.05 mg/kg to about 2 mg/kg,from about 1 mg/kg to about 5 mg/kg, from about 2 mg/kg to about 10mg/kg, from about 4 mg/kg to about 16 mg/kg, from about 5 mg/kg to about20 mg/kg, from about 8 mg/kg to about 24 mg/kg, from about 10 mg/kg toabout 30 mg/kg, from about 12 mg/kg to about 32 mg/kg, from about 14mg/kg to about 34 mg/kg, from about 16 mg/kg to about 36 mg/kg, fromabout 18 mg/kg to about 38 mg/kg, from about 20 mg/kg to about 40 mg/kg,from about 22 mg/kg to about 42 mg/kg, from about 24 mg/kg to about 44mg/kg, from about 26 mg/kg to about 46 mg/kg, from about 28 mg/kg toabout 48 mg/kg, from about 30 mg/kg to about 50 mg/kg, from about 35mg/kg to about 55 mg/kg, from about 40 mg/kg to about 60 mg/kg, fromabout 45 mg/kg to about 65 mg/kg, from about 50 mg/kg to about 75 mg/kg,from about 60 mg/kg to about 240 mg/kg, from about 70 mg/kg to about 350mg/kg, from about 80 mg/kg to about 400 mg/kg, from about 90 mg/kg toabout 450 mg/kg, from about 100 mg/kg to about 500 mg/kg, from about0.01 g/kg to about 1 g/kg, from about 0.05 g/kg to about 2 g/kg, fromabout 1 g/kg to about 5 g/kg, or more than 5 g/kg.

In some embodiments, pharmaceutical compositions that are or includeSBPs may be administered at a dose sufficient to yield desiredtherapeutic agent or SBP concentration levels in subject tissue orfluids (e.g., blood, plasma, urine, etc.). In some embodiments, dosesare adjusted to achieve subject therapeutic agent or SBP concentrationlevels in subject tissues or fluids of from about 1 pg/mL to about 5pg/mL, from about 2 pg/mL to about 10 pg/mL, from about 4 pg/mL to about16 pg/mL, from about 5 pg/mL to about 20 pg/mL, from about 8 pg/mL toabout 24 pg/mL, from about 10 pg/mL to about 30 pg/mL, from about 12pg/mL to about 32 pg/mL, from about 14 pg/mL to about 34 pg/mL, fromabout 16 pg/mL to about 36 pg/mL, from about 18 pg/mL to about 38 pg/mL,from about 20 pg/mL to about 40 pg/mL, from about 22 pg/mL to about 42pg/mL, from about 24 pg/mL to about 44 pg/mL, from about 26 pg/mL toabout 46 pg/mL, from about 28 pg/mL to about 48 pg/mL, from about 30pg/mL to about 50 pg/mL, from about 35 pg/mL to about 55 pg/mL, fromabout 40 pg/mL to about 60 pg/mL, from about 45 pg/mL to about 65 pg/mL,from about 50 pg/mL to about 75 pg/mL, from about 60 pg/mL to about 240pg/mL, from about 70 pg/mL to about 350 pg/mL, from about 80 pg/mL toabout 400 pg/mL, from about 90 pg/mL to about 450 pg/mL, from about 100pg/mL to about 500 pg/mL, from about 0.01 ng/mL to about 1 ng/mL, fromabout 0.05 ng/mL to about 2 ng/mL, from about 1 ng/mL to about 5 ng/mL,from about 2 ng/mL to about 10 ng/mL, from about 4 ng/mL to about 16ng/mL, from about 5 ng/mL to about 20 ng/mL, from about 8 ng/mL to about24 ng/mL, from about 10 ng/mL to about 30 ng/mL, from about 12 ng/mL toabout 32 ng/mL, from about 14 ng/mL to about 34 ng/mL, from about 16ng/mL to about 36 ng/mL, from about 18 ng/mL to about 38 ng/mL, fromabout 20 ng/mL to about 40 ng/mL, from about 22 ng/mL to about 42 ng/mL,from about 24 ng/mL to about 44 ng/mL, from about 26 ng/mL to about 46ng/mL, from about 28 ng/mL to about 48 ng/mL, from about 30 ng/mL toabout 50 ng/mL, from about 35 ng/mL to about 55 ng/mL, from about 40ng/mL to about 60 ng/mL, from about 45 ng/mL to about 65 ng/mL, fromabout 50 ng/mL to about 75 ng/mL, from about 60 ng/mL to about 240ng/mL, from about 70 ng/mL to about 350 ng/mL, from about 80 ng/mL toabout 400 ng/mL, from about 90 ng/mL to about 450 ng/mL, from about 100ng/mL to about 500 ng/mL, from about 0.01 μg/mL to about 1 μg/mL, fromabout 0.05 μg/mL to about 2 μg/mL, from about 1 μg/mL to about 5 μg/mL,from about 2 μg/mL to about 10 μg/mL, from about 4 μg/mL to about 16μg/mL, from about 5 μg/mL to about 20 μg/mL, from about 8 μg/mL to about24 μg/mL, from about 10 μg/mL to about 30 μg/mL, from about 12 μg/mL toabout 32 μg/mL, from about 14 μg/mL to about 34 μg/mL, from about 16μg/mL to about 36 μg/mL, from about 18 μg/mL to about 38 μg/mL, fromabout 20 μg/mL to about 40 μg/mL, from about 22 μg/mL to about 42 μg/mL,from about 24 μg/mL to about 44 μg/mL, from about 26 μg/mL to about 46μg/mL, from about 28 μg/mL to about 48 μg/mL, from about 30 μg/mL toabout 50 μg/mL, from about 35 μg/mL to about 55 μg/mL, from about 40μg/mL to about 60 μg/mL, from about 45 μg/mL to about 65 μg/mL, fromabout 50 μg/mL to about 75 μg/mL, from about 60 μg/mL to about 240μg/mL, from about 70 μg/mL to about 350 μg/mL, from about 80 μg/mL toabout 400 μg/mL, from about 90 μg/mL to about 450 μg/mL, from about 100μg/mL to about 500 μg/mL, from about 0.01 mg/mL to about 1 mg/mL, fromabout 0.05 mg/mL to about 2 mg/mL, from about 1 mg/mL to about 5 mg/mL,from about 2 mg/mL to about 10 mg/mL, from about 4 mg/mL to about 16mg/mL, from about 5 mg/mL to about 20 mg/mL, from about 8 mg/mL to about24 mg/mL, from about 10 mg/mL to about 30 mg/mL, from about 12 mg/mL toabout 32 mg/mL, from about 14 mg/mL to about 34 mg/mL, from about 16mg/mL to about 36 mg/mL, from about 18 mg/mL to about 38 mg/mL, fromabout 20 mg/mL to about 40 mg/mL, from about 22 mg/mL to about 42 mg/mL,from about 24 mg/mL to about 44 mg/mL, from about 26 mg/mL to about 46mg/mL, from about 28 mg/mL to about 48 mg/mL, from about 30 mg/mL toabout 50 mg/mL, from about 35 mg/mL to about 55 mg/mL, from about 40mg/mL to about 60 mg/mL, from about 45 mg/mL to about 65 mg/mL, fromabout 50 mg/mL to about 75 mg/mL, from about 60 mg/mL to about 240mg/mL, from about 70 mg/mL to about 350 mg/mL, from about 80 mg/mL toabout 400 mg/mL, from about 90 mg/mL to about 450 mg/mL, from about 100mg/mL to about 500 mg/mL, from about 0.01 g/mL to about 1 g/mL.

In some embodiments, pharmaceutical compositions that are or includeSBPs are provided in one or more doses and are administered one or moretimes to subjects. Some pharmaceutical compositions are provided in onlya single administration. Some pharmaceutical compositions are providedaccording to a dosing schedule that include two or more administrations.Each administration may be at the same dose or may be different from aprevious and/or subsequent dose. In some embodiments, subjects areprovided an initial dose that is higher than subsequent doses (referredto herein as a “loading dose”). In some embodiments, doses are decreasedover the course of administration. In some embodiments, dosing schedulesinclude pharmaceutical composition administration from about every 2hours to about every 10 hours, from about every 4 hours to about every20 hours, from about every 6 hours to about every 30 hours, from aboutevery 8 hours to about every 40 hours, from about every 10 hours toabout every 50 hours, from about every 12 hours to about every 60 hours,from about every 14 hours to about every 70 hours, from about every 16hours to about every 80 hours, from about every 18 hours to about every90 hours, from about every 20 hours to about every 100 hours, from aboutevery 22 hours to about every 120 hours, from about every 24 hours toabout every 132 hours, from about every 30 hours to about every 144hours, from about every 36 hours to about every 156 hours, from aboutevery 48 hours to about every 168 hours, from about every 2 days toabout every 10 days, from about every 4 days to about every 15 days,from about every 6 days to about every 20 days, from about every 8 daysto about every 25 days, from about every 10 days to about every 30 days,from about every 12 days to about every 35 days, from about every 14days to about every 40 days, from about every 16 days to about every 45days, from about every 18 days to about every 50 days, from about every20 days to about every 55 days, from about every 22 days to about every60 days, from about every 24 days to about every 65 days, from aboutevery 30 days to about every 70 days, from about every 2 weeks to aboutevery 8 weeks, from about every 3 weeks to about every 12 weeks, fromabout every 4 weeks to about every 16 weeks, from about every 5 weeks toabout every 20 weeks, from about every 6 weeks to about every 24 weeks,from about every 7 weeks to about every 28 weeks, from about every 8weeks to about every 32 weeks, from about every 9 weeks to about every36 weeks, from about every 10 weeks to about every 40 weeks, from aboutevery 11 weeks to about every 44 weeks, from about every 12 weeks toabout every 48 weeks, from about every 14 weeks to about every 52 weeks,from about every 16 weeks to about every 56 weeks, from about every 20weeks to about every 60 weeks, from about every 2 months to about every6 months, from about every 3 months to about every 12 months, from aboutevery 4 months to about every 18 months, from about every 5 months toabout every 24 months, from about every 6 months to about every 30months, from about every 7 months to about every 36 months, from aboutevery 8 months to about every 42 months, from about every 9 months toabout every 48 months, from about every 10 months to about every 54months, from about every 11 months to about every 60 months, from aboutevery 12 months to about every 66 months, from about 2 years to about 5years, from about 3 years to about 10 years, from about 4 years to about15 years, from about 5 years to about 20 years, from about 6 years toabout 25 years, from about 7 years to about 30 years, from about 8 yearsto about 35 years, from about 9 years to about 40 years, from about 10years to about 45 years, from about 15 years to about 50 years, or morethan every 50 years.

In some embodiments, pharmaceutical compositions that are or includeSBPs may be administered at a dose sufficient to provide atherapeutically effective amount of therapeutic agents or SBPs. As usedherein, the term “therapeutically effective amount” refers to an amountof an agent sufficient to achieve a therapeutically effective outcome.As used herein, the term “therapeutically effective outcome” refers to aresult of treatment where at least one objective of treatment is met. Insome embodiments, a therapeutically effective amount is provided in asingle dose. In some embodiments, a therapeutically effective amount isadministered according to a dosing schedule that includes a plurality ofdoses. Those skilled in the art will appreciate that in someembodiments, a unit dosage form may be considered to include atherapeutically effective amount of a particular agent or entity if itincludes an amount that is effective when administered as part of such adosage regimen.

Administration

In some embodiments, pharmaceutical compositions that are or includeSBPs may be administered according to one or more administration routes.In some embodiments, administration is transdermal, intravenous bolus,intralesional (within or introduced directly to a localized lesion),intraocular (within the eye), intracartilaginous (within a cartilage),insufflation (snorting), intravascular (within a vessel or vessels),buccal (directed toward the cheek), percutaneous, submucosal, cutaneous,epicutaneous (application onto the skin), intramedullary (within themarrow cavity of a bone), intramuscular (into a muscle), subcutaneous(under the skin), intragastric (within the stomach), nasaladministration (through the nose), endosinusial, soft tissue,subconjunctival, oral (by way of the mouth), periodontal, periarticular,auricular (in or by way of the ear), intratubular (within the tubules ofan organ), intradermal (into the skin itself), intravitreal (through theeye), irrigation (to bathe or flush open wounds or body cavities), inear drops, endotracheal, intraosseous infusion (into the bone marrow),caudal block, intra-articular, intracomeal (within the cornea),extracorporeal, transmucosal (diffusion through a mucous membrane),topical, oropharyngeal (directly to the mouth and pharynx), occlusivedressing technique (topical route administration which is then coveredby a dressing which occludes the area), intraarterial (into an artery),intrasinal (within the nasal or periorbital sinuses), intraductal(within a duct of a gland), transdermal (diffusion through the intactskin for systemic distribution), retrobulbar (behind the pons or behindthe eyeball), intravenous (into a vein), intrasynovial (within thesynovial cavity of a joint), intratumor (within a tumor), eye drops(onto the conjunctiva), respiratory (within the respiratory tract byinhaling orally or nasally for local or systemic effect), and/orophthalmic (to the external eye).

In some embodiments, pharmaceutical compositions that are or includeSBPs may be administered by intravitreal administration, intraretinaladministration, intracorneal administration, intrascleraladministration, punctal administration, administration to the anteriorsub-Tenon's, suprachoroidal administration, administration to theposterior sub-Tenon's, subretinal administration, administration to thefomix, administration to the lens, intra-aqueous humor administration,transmucosal administration, transdermal administration, soft tissueadministration, subcutaneous administration, topical administration,insufflation, enema, eye drops, ear drops, or intravesical infusion. Insome embodiments, the SBPs described herein may be administered viainjection. Injection site reactions may be monitored via any methodknown to one skilled in the art.

In some embodiments, SBPs may be administered for localized treatment(e.g., see United States Publication Numbers US20170368236 andUS20110171239, the contents of each of which are herein incorporated byreference in their entirety). In some embodiments, SBPs may beadministered for treatment of areas located further away fromadministration sites (e.g., see Aykac et 71. (2017) Genes0378-1119(17)30868-30865, the contents of which are herein incorporatedby reference in their entirety).

In some embodiments, administration includes ocular administration. Asused herein, the term “ocular administration” refers to delivery of anagent to an eye. Ocular administration may include, but is not limitedto, topical administration (e.g., using eye drops, ointments, orcreams), intraocular administration, intravitreal administration,intraretinal administration, intracomeal administration, intrascleraladministration, punctal administration, administration to the anteriorsub-Tenon's, suprachroidal administration, administration to theposterior sub-Tenon's, subretinal administration, administration to thefomix, administration to the lens, administration to the anteriorsegment, administration to the posterior segment, macularadministration, and intra-aqueous humor administration. Administrationmay include intravitreal injection. In some embodiments, intraocular SBPadministration reduces intraocular pressure.

In some embodiments, SBPs described herein may be administered using anyform of injection device, for example a syringe/needle device of a gaugesuitable for the application. As a non-limiting example, SBPs may beadministered using a syringe/needle with a gauge of 6, 6.5, 7, 7.5, 8,8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5,16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5,23, 23.5, 24, 24.5, 25, 25.5, 26, 27, 28, 29, 30, 31, 32, 33, or 34. Insome embodiments the administration is intravitreal using a 22-gaugeneedle. In some embodiments, the administration is intravitreal using a27-gauge needle.

In some embodiments, the SBP is formatted as hydrogels and administeredusing a needle with a gauge of at least 27. For example, SBP hydrogelsmay be administered using a needle with a gauge of 27, 28, 29, 30, 31,32, 33, or 34. In one embodiment, SBP hydrogels may be administeredusing a 27-gauge needle.

In some embodiments, the SBP is formatted as rods and administered usinga needle with a gauge of at least 20. For example, SBP rods may beadministered using needles with a gauge of 20, 20.5, 21, 21.5, 22, 22.5,23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 28, 29, 30, or more than 30.In one embodiment, SBP rods are administered using a 21-gauge needle. Inone embodiment, SBP rods are administered using a 22-gauge needle.

In some embodiments, SBP administration does not result in aninflammatory response beyond transient local foreign body reaction. Insome embodiments, SBPs are biocompatible. In some embodiments, SBPs arewell tolerated after administration. In some embodiments, SBPs are welltolerated after intravitreal administration.

In some embodiments, intraocular SBP administration provides continuousdelivery of therapeutic agents. In some embodiments, the intraocular SBPadministration results in detectable levels of ocular therapeutic agentin vitreous humor. Ocular therapeutic agents may be detectable invitreous humor for from about 1 day to about 60 days, from about 1 weekto about 24 weeks, from about 1 month to about 6 months, from about 3months to about 24 months, from about 1 year to about 5 years, or formore than 5 years. Ocular therapeutic agent levels may steady over anyof such periods of time.

In some embodiments, SBP administration or SBP-based therapeutic agentadministration occurs over a period of time, referred to herein as the“administration period.” During administration periods, administrationmay be continuous or may be separated into two or more administrations.In some embodiments, administration periods may be from about 1 min toabout 30 min, from about 10 min to about 45 min, from about 20 min toabout 60 min, from about 40 min to about 90 min, from about 2 hours toabout 10 hours, from about 4 hours to about 20 hours, from about 6 hoursto about 30 hours, from about 8 hours to about 40 hours, from about 10hours to about 50 hours, from about 12 hours to about 60 hours, fromabout 14 hours to about 70 hours, from about 16 hours to about 80 hours,from about 18 hours to about 90 hours, from about 20 hours to about 100hours, from about 22 hours to about 120 hours, from about 24 hours toabout 132 hours, from about 30 hours to about 144 hours, from about 36hours to about 156 hours, from about 48 hours to about 168 hours, fromabout 2 days to about 10 days, from about 4 days to about 15 days, fromabout 6 days to about 20 days, from about 8 days to about 25 days, fromabout 10 days to about 30 days, from about 12 days to about 35 days,from about 14 days to about 40 days, from about 16 days to about 45days, from about 18 days to about 50 days, from about 20 days to about55 days, from about 22 days to about 60 days, from about 24 days toabout 65 days, from about 30 days to about 70 days, from about 2 weeksto about 8 weeks, from about 3 weeks to about 12 weeks, from about 4weeks to about 16 weeks, from about 5 weeks to about 20 weeks, fromabout 6 weeks to about 24 weeks, from about 7 weeks to about 28 weeks,from about 8 weeks to about 32 weeks, from about 9 weeks to about 36weeks, from about 10 weeks to about 40 weeks, from about 11 weeks toabout 44 weeks, from about 12 weeks to about 48 weeks, from about 14weeks to about 52 weeks, from about 16 weeks to about 56 weeks, fromabout 20 weeks to about 60 weeks, from about 2 months to about 6 months,from about 3 months to about 12 months, from about 4 months to about 18months, from about 5 months to about 24 months, from about 6 months toabout 30 months, from about 7 months to about 36 months, from about 8months to about 42 months, from about 9 months to about 48 months, fromabout 10 months to about 54 months, from about 11 months to about 60months, from about 12 months to about 66 months, from about 2 years toabout 5 years, from about 3 years to about 10 years, from about 4 yearsto about 15 years, from about 5 years to about 20 years, from about 6years to about 25 years, from about 7 years to about 30 years, fromabout 8 years to about 35 years, from about 9 years to about 40 years,from about 10 years to about 45 years, from about 15 years to about 50years, or more than 50 years.

Depot Administration

In some embodiments, SBPs may be administered by or be used toadminister therapeutic agents by depot administration. As used herein,the term “depot” refers to a concentration of one or more agents in aparticular region or in association with a composition or device. Withdepot administration, the one or more agents exit or diffuse from theconcentration into surrounding areas. Agents administered by depotadministration may be SBPs. In some embodiments, SBPs are depots fortherapeutic agents, wherein the therapeutic agents exit or diffuse fromthe SBPs. In some embodiments, the SBPs may be utilized for the localdelivery of therapeutic agents. In some embodiments, depots areimplants. In some embodiments, depots are gels or hydrogels. In someembodiments, depot administration of an SBP may reduce the number oftimes a therapeutic agent needs to be administered. In some embodiments,depot administration of an SBP may replace oral administration of atherapeutic agent.

Controlled Release

In some embodiments, SBPs and related methods described herein be may beused for controlled release of therapeutic agents. As used herein, theterm “controlled release” refers to regulated movement of factors fromspecific locations to surrounding areas. In some embodiments, thespecific location is a depot. Controlled release of factors from depotsmay be regulated by interactions between therapeutic agents and depotcomponents. Such interactions may, for example, modulate therapeuticagent diffusion rate and/or affect therapeutic agent stability and/ordegradation. In some embodiments, the depot is an SBP. In someembodiments, factors subject to controlled release from depots are SBPs.In some embodiments, therapeutic agents are subject to controlledrelease from SBP depots.

In some embodiments, SBPs may control payload release by extendingpayload half-life. As used herein, the term “half-life” refers to thelength of time necessary for levels of a factor to be reduced (e.g.,through clearance or degradation) by 50%. Some payloads may exhibitshortened half-life in water (e.g., due to hydrolysis). SBPs may protectpayloads from exposure to water, thereby improving payload half-life. Insome embodiments, methods of increasing payload half-life using SBPs mayinclude any of those described in United States PublicationUS20100028451, the contents of which are herein incorporated byreference in their entirety. Methods of improving payload half-life maybe carried out in vitro or in vivo. In some embodiments, SBP-basedmethods of improving payload half-life may enable therapeutic indicationtreatment with fewer doses and/or treatments. Such methods may includeany of those described in International Publication Number WO2017139684,the contents of which are herein incorporated by reference in theirentirety. In some embodiments, payload half-life may be extended by fromabout 0.01% to about 1%, from about 0.05% to about 2%, from about 1% toabout 5%, from about 2% to about 10%, from about 3% to about 15%, fromabout 4% to about 20%, from about 5% to about 25%, from about 6% toabout 30%, from about 7% to about 35%, from about 8% to about 40%, fromabout 9% to about 45%, from about 10% to about 50%, from about 12% toabout 55%, from about 14% to about 60%, from about 16% to about 65%,from about 18% to about 70%, from about 20% to about 75%, from about 22%to about 80%, from about 24% to about 85%, from about 26% to about 90%,from about 28% to about 95%, from about 30% to about 100%, from about32% to about 105%, from about 34% to about 110%, from about 36% to about115%, from about 38% to about 120%, from about 40% to about 125%, fromabout 42% to about 130%, from about 44% to about 135%, from about 46% toabout 140%, from about 48% to about 145%, from about 50% to about 150%,from about 60% to about 175%, from about 70% to about 200%, from about80% to about 225%, from about 90% to about 250%, from about 100% toabout 275%, from about 110% to about 300%, from about 120% to about325%, from about 130% to about 350%, from about 140% to about 375%, fromabout 150% to about 400%, from about 170% to about 450%, from about 190%to about 500%, from about 210% to about 550%, from about 230% to about600%, from about 250% to about 650%, from about 270% to about 700%, fromabout 290% to about 750%, from about 310% to about 800%, from about 330%to about 850%, from about 350% to about 900%, from about 370% to about950%, from about 390% to about 1000%, from about 410% to about 1050%,from about 430% to about 1100%, from about 450% to about 1500%, fromabout 480% to about 2000%, from about 510% to about 2500%, from about540% to about 3000%, from about 570% to about 3500%, from about 600% toabout 4000%, from about 630% to about 4500%, from about 660% to about5000%, from about 690% to about 5500%, from about 720% to about 6000%,from about 750% to about 6500%, from about 780% to about 7000%, fromabout 810% to about 7500%, from about 840% to about 8000%, from about870% to about 8500%, from about 900% to about 9000%, from about 930% toabout 9500%, from about 960% to about 10000%, or more than 10000%.

In some embodiments, SBP depots may be used for controlled release oftherapeutic agents, wherein release is facilitated by diffusion. Suchmethods may include any of those described in United States PublicationNumber US20170333351, the contents of which are herein incorporated byreference in their entirety. Therapeutic agent diffusion may be slowed(i.e., controlled) by SBP depots leading to extended release periods.Extended therapeutic agent release periods may enable longeradministration periods. In some embodiments, administration periods areextended by from about 0.01% to about 1%, from about 0.05% to about 2%,from about 1% to about 5%, from about 2% to about 10%, from about 3% toabout 15%, from about 4% to about 20%, from about 5% to about 25%, fromabout 6% to about 30%, from about 7% to about 35%, from about 8% toabout 40%, from about 9% to about 45%, from about 10% to about 50%, fromabout 12% to about 55%, from about 14% to about 60%, from about 16% toabout 65%, from about 18% to about 70%, from about 20% to about 75%,from about 22% to about 80%, from about 24% to about 85%, from about 26%to about 90%, from about 28% to about 95%, from about 30% to about 100%,from about 32% to about 105%, from about 34% to about 110%, from about36% to about 115%, from about 38% to about 120%, from about 40% to about125%, from about 42% to about 130%, from about 44% to about 135%, fromabout 46% to about 140%, from about 48% to about 145%, from about 50% toabout 150%, from about 60% to about 175%, from about 70% to about 200%,from about 80% to about 225%, from about 90% to about 250%, from about100% to about 275%, from about 110% to about 300%, from about 120% toabout 325%, from about 130% to about 350%, from about 140% to about375%, from about 150% to about 400%, from about 170% to about 450%, fromabout 190% to about 500%, from about 210% to about 550%, from about 230%to about 600%, from about 250% to about 650%, from about 270% to about700%, from about 290% to about 750%, from about 310% to about 800%, fromabout 330% to about 850%, from about 350% to about 900%, from about 370%to about 950%, from about 390% to about 1000%, from about 410% to about1050%, from about 430% to about 1100%, from about 450% to about 1500%,from about 480% to about 2000%, from about 510% to about 2500%, fromabout 540% to about 3000%, from about 570% to about 3500%, from about600% to about 4000%, from about 630% to about 4500%, from about 660% toabout 5000%, from about 690% to about 5500%, from about 720% to about6000%, from about 750% to about 6500%, from about 780% to about 7000%,from about 810% to about 7500%, from about 840% to about 8000%, fromabout 870% to about 8500%, from about 900% to about 9000%, from about930% to about 9500%, from about 960% to about 10000%,

In some embodiments, the controlled release of a therapeutic agent forthe treatment of a condition, disease, or indication may be facilitatedby the degradation and/or dissolution of SBPs. Such methods may becarried according to those described in International PublicationNumbers WO2013126799, WO2017165922, and U.S. Pat. No. 8,530,625, thecontents of each of which are herein incorporated by reference in theirentirety. SBP degradation and/or dissolution may expose increasingamounts of therapeutic agents over time for treatment of therapeuticindications.

In some embodiments, therapeutic agent release from SBPs may bemonitored via high performance liquid chromatography (HPLC),ultra-performance liquid chromatography (UPLC), and/or other methodsknown to those skilled in the art.

SBP hydrogels may be used to extend payload release periods (e.g., asshown for extended release of small molecule in InternationalPublication Number WO2017139684, the contents of which are hereinincorporated by reference in their entirety. In some embodiments, SBPhydrogels are used to provide extended release of therapeutic agents(e.g., biological agents). Hydrogel networks may stabilize such agentsand support their release as the hydrogel degrades. This effect servesto extend agent release and may be modulated by varying factorsincluding processed silk molecular weight, concentration, excipienttype, pH, and temperature. In some embodiments, processed silk molecularweight, concentration, excipient type, pH, and processing temperatureused to prepare SBPs may be modulated to achieve desired payload releaseperiods for specific therapeutic agents.

In some embodiments, SBPs may be lyophilized together with therapeuticagents. In some embodiments, combined lyophilization may induce furtherinteractions between therapeutic agents and SBPs. These interactions maybe maintained through SBP preparation and support extended payloadrelease. Payload release may be dependent on SBP degradation and/ordissolution. In some embodiments, SBP p-sheet content is increased(e.g., via water annealing), thereby increasing SBP insolubility inwater. Such SBPs may exhibit increased payload release periods. In someembodiments, these SBPs may include therapeutic agent stabilizingproperties to extend administration periods and/or therapeutic agenthalf-life.

In some embodiments, SBPs described herein maintain and/or improve thecontrolled delivery of a therapeutic agent. In some embodiments, SBPslengthen payload release period and/or administration period by at least1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9hours, at least 10 hours, at least 11 hours, at least 12 hours, at least13 hours, at least 14 hours, at least 15 hours, at least 16 hours, atleast 17 hours, at least 18 hours, at least 19 hours, at least 20 hours,at least 21 hours, at least 22 hours, at least 23 hours, or at least 24hours. In some embodiments, SBPs lengthen payload release period and/oradministration period by at least 1 day, at least 2 days, at least 3days, at least 4 days, at least 5 days, at least 6 days, at least 7days, at least 8 days, at least 9 days, at least 10 days, at least 11days, at least 12 days, at least 13 days, at least 2 weeks, at least 3weeks, at least 1 month, at least 6 weeks, at least 2 months, at least10 weeks, at least 3 months, at least 6 months, at least 9 months, or atleast 1 year.

In some embodiments, SBPs may be used to modulate depot release oftherapeutic agents. Some SBPs may release therapeutic agents accordingto near zero-order kinetics. In some embodiments, SBPs may releasetherapeutic agents according to first-order kinetics. In someembodiments, therapeutic agent release rate may be modulated bypreparing SBP depots with modification of one or more of density,loading, drying method, silk fibroin molecular weight, and silk fibroinconcentration.

In some embodiments, SBPs are prepared to release from about 0.01% toabout 1%, from about 0.05% to about 2%, from about 1% to about 5%, fromabout 2% to about 10%, from about 3% to about 15%, from about 4% toabout 20%, from about 5% to about 25%, from about 6% to about 30%, fromabout 7% to about 35%, from about 8% to about 40%, from about 9% toabout 45%, from about 10% to about 50%, from about 12% to about 55%,from about 14% to about 60%, from about 16% to about 65%, from about 18%to about 70%, from about 20% to about 75%, from about 22% to about 80%,from about 24% to about 85%, from about 26% to about 90% from about 28%to about 95%, from about 30% to about 100% of the total amount oftherapeutic or macromolecular therapeutic agent to be delivered.

In some embodiments, the SBPs (e.g. hydrogels) demonstrate a sustainedrelease of a therapeutic agent, with near steady state concentrations.In some embodiments, the sustained release is at a level at or near theeffective concentration. In some embodiments, the sustained release isat greater than or equal to the effective concentration. In someembodiments the effective concentration is the IC₅₀, the EC₅₀, or theEC₈₀.

Delivery

SBPs may be delivered to cells, tissues, organs and/or organisms innaked form. As used herein in, “naked” delivery refers to delivery of anactive agent with minimal or with no additional formulation ormodification. Naked SBPs may be delivered to cells, tissues, organsand/or organisms using routes of administration known in the art anddescribed herein. In some embodiments, naked delivery may includeformulation in a simple buffer such as saline, phosphate buffer, or PBS.

In some embodiments, SBPs may be prepared with one or more cellpenetration agents, pharmaceutically acceptable carriers, deliveryagents, bioerodible or biocompatible polymers, solvents, and/orsustained-release delivery depots. SBPs may be delivered to cells usingroutes of administration known in the art and described herein. In someembodiments. SBPs may be formulated for direct delivery to organs ortissues in any of several ways in the art including, but not limited to,direct soaking or bathing, via a catheter, by gels, powder, ointments,creams, gels, lotions, and/or drops, or by using substrates (e.g.,fabric or biodegradable materials) coated or impregnated with SBPs.

Detectable Agents and Labels

In some embodiments, SBPs described herein may be formulated withdetectable labels. As used herein, the term “detectable label” refers toany incorporated compound or entity that facilitates some form ofidentification. Detectable labels may include, but are not limited tovarious organic small molecules, inorganic compounds, nanoparticles,enzymes or enzyme substrates, fluorescent materials, luminescentmaterials (e.g., luminol), bioluminescent materials (e.g., luciferase,luciferin, and aequorin), chemiluminescent materials, radioactivematerials (e.g., ¹⁸F, ⁶⁷Ga, ⁸¹mKr, ⁸²Rb, ¹¹¹In, ¹²²I, ¹³³Xe, ²⁰¹Tl,¹²⁵I, ³⁵S, ¹⁴C, ³H, or ⁹⁹mTc (e.g., as pertechnetate (technetate(VII),TcO⁴⁻)), contrast agents (e.g., gold, gold nanoparticles, gadolinium,chelated Gd, iron oxides, superparamagnetic iron oxide (SPIO),monocrystalline iron oxide nanoparticles (MIONs), and ultrasmallsuperparamagnetic iron oxide (USPIO)), manganese chelates (e.g.,Mn-DPDP), barium sulfate, iodinated contrast media (iohexol),microbubbles, or perfluorocarbons). Such optically-detectable labelsinclude for example, without limitation,4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridine andderivatives (e.g., acridine and acridine isothiocyanate);5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS);4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate;N-(4-anilino-1-naphthyl)maleimide; anthranilamide; BODIPY; BrilliantYellow; coumarin and derivatives (e.g., coumarin,7-amino-4-methylcoumarin (AMC, Coumarin 120), and7-amino-4-trifluoromethylcoumarin (Coumarin 151)); cyanine dyes;cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI); 5′5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]-naphthalene-1-sulfonyl chloride (DNS, dansylchloride);4-dimethvlaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives (e.g., eosin and eosin isothiocyanate); erythrosin andderivatives (e.g., erythrosin B and erythrosin isothiocyanate);ethidium; fluorescein and derivatives (e.g., 5-carboxyfluorescein (FAM),5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein, fluorescein,fluorescein isothiocyanate, X-rhodamine-5-(and-6)-isothiocyanate (QFITCor XRITC), and fluorescamine);2-[2-[3-[[1,3-dihydro-1,1-dimethyl-3-(3-sulfopropyl)-2H-benz[e]indol-2-ylidene]ethylidene]-2-[4-(ethoxycarbonyl)-1-piperazinyl]-1-cyclopenten-1-yl]ethenyl]-1,1-dimethyl-3-(3-sulforpropyl)-1H-benz[e]indoliumhydroxide, inner salt, compound with n,ndiethylethanamine(1:1) (IR144);5-chloro-2-[2-[3-[(5-chloro-3-ethyl-2(3H)-benzothiazolylidene)ethylidene]-2-(diphenylamino)-1-cyclopenten-1-yl]ethenyl]-3-ethylbenzothiazolium perchlorate (IR140); Malachite Green isothiocyanate;4-methylumbelliferone orthocresolphthalein; nitrotyrosine;pararosaniline; Phenol Red; B-phycocrythrin; o-phthaldialdehyde; pyreneand derivatives(e.g., pyrene, pyrene butyrate, and succinimidyl1-pyrene); butyrate quantum dots; Reactive Red 4 (CIBACRON™ BrilliantRed 3B-A); rhodamine and derivatives (e.g., 6-carboxy-Xrhodamine (ROX),6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloriderhodarnine (Rhod), rhodamine B, rhodamine 123, rhodamine Xisothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloridederivative of sulforhodamine 101 (Texas Red),N,N,N′,N′tetramethyl-6-carboxyrhodamine (TAMRA) tetramethyl rhodamine,and tetramethyl rhodamine isothiocyanate (TRITC)); riboflavin; rosolicacid; terbium chelate derivatives; Cyanine-3 (Cy3); Cyanine-5 (Cy5);cyanine-5.5 (C5.5), Cyanine-7 (Cy7); IRD 700; IRD 800; Alexa 647; LaJolta Blue; phthalo cyanine; and naphthalo cyanine.

In some embodiments, the detectable labels may include non-detectableprecursors that becomes detectable upon activation (e.g., fluorogenictetrazine-fluorophore constructs, tetrazine-BODIPY FL, tetrazine-OregonGreen 488, or tetrazine-BODIPY TMR-X) or enzyme activatable fluorogenicagents (e.g., PROSENSE® (VisEn Medical)). In vitro assays in whichenzyme labeled compositions can be used include, but are not limited to,enzyme linked immunosorbent assays (ELISAs), immunoprecipitation assays,immunofluorescence, enzyme immunoassays (EIA), radioimmunoassays (RIA),and Western blot analysis.

Therapeutic Devices

In some embodiments, SBPs may be or may be included in therapeuticdevices. In some embodiments, therapeutic devices may be coated withSBPs described herein. Some therapeutic devices may include therapeuticagents. In some embodiments, the use of SBPs within therapeutic devicesmay enable the delivery of therapeutic agents via such therapeuticdevices. Some therapeutic devices may include synthetic materials. Insome embodiments, therapeutic devices include, but are not limited to,artificial blood vessels, artificial liver, artificial organ, bandage,breast augmentation, cartilage replacement, ear drum repair, filler,hemostatic sponge, implant, silk contact lens, stem cell, surgical mesh,surgical suture, tissue replacement, vascular patch, wound dressing,antenna, applier, artificial heart, artificial heart valve, assembly,balloon, barrier, biosensor, biotransducer, breast implant, cableassembly, caliper, capacitor, carrier, clamp, cochlear implant,connector, comeal implant, coronary stent, cryotome, degradable device,delivery device, dental implant, dermatome, detector, diagnostic device,dilator, diode, discharge device, display technology, distractor, drillbit, electronic device, gastric stimulator, graft, grasper, harmonicscalpel, hemostatic device, imaging apparatus, implant, implant forcontinuous drug delivery, implantable cardioverter-defibrillator,integrated circuit, intraocular lens, intrauterine device, lancet,LIGASURE™, liner, magnetic or inductive device, magnetic resonanceimaging apparatus, mechanical assembly, medical device, memristor,module, needle, nerve stimulator, network, neurostimulator, occluder,optoelectronic device, pacemaker, patch, pen, piezoelectric device, pin,pipe, plate, positioner, power source, probe, prosthesis, prosthetic,protection device, removable device, resistor, retractor, rod, rongeur,rope, ruler, scalpel, scope, screw, semiconductor, sensor, solution,specula, stent, stent, sterotactic device, suction tip, suction tube,surgical device, surgical mesh, surgical scissor, surgical staple,suture, switch, temperature sensor, terminal, tie, tip, transducer,transistor, tube, tympanostomy tube, ultrasound tissue disruptor, vacuumtube, vacuum valve, ventilation system, water balloon, wire, bleb, gel,gel that hardens after implantation, implant, lacrimal plug, lens, plug,punctal plug, rod, slurry, slurry that hardens after implantation, andsolids.

In some embodiments, therapeutic devices include implants. As usedherein, the term “implant” refers to a device that may be embedded in orwithin a carrier. Implants used in therapeutic applications aretypically embedded in subjects to support, repair, replace, or enhanceone or more tissues or features. In some embodiments, implants includeone or more excipients and/or one or more therapeutic agents (e.g., anyof the excipients or therapeutic agents presented herein. Implants mayinclude depots for therapeutic agent release, as described herein. Insome embodiments, implants may include one or more coatings, gels,hydrogels, scaffolds, particles, or therapeutic devices (e.g., any ofthose listed above).

Some implants may be prepared by mixing a therapeutic agent with aprocessed silk solution. The solution may be heated to form thehydrogel. Some hydrogels may be heated to dryness and some hydrogels maybe frozen and lyophilized to form an implant. Further, implants may becompressed to slow hydration as well as to slow the release oftherapeutic agent. Excipients may be incorporated into processed silksolutions prior to hydrogel formation to allow for scaffold formationduring the freezing/lyophilization process. Excipients may includegelling agents such as, but are not limited to, poloxamers, PEG's,mannitol, sorbitol, etc. Rods or scaffolds may be formed from hydrogelsby compression or extrusion. The rods may be formed taking intoconsideration the dimensions and/or properties that allow for injectionthrough small gauge needles (e.g., with a gauge of more than 20). Asnon-limiting examples, SBP rods may be injectable through needles with agauge of 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26,26.5, 27, 28, 29, 30, or more than 30. In one embodiment, SBP rods areinjectable with a 21-gauge needle. In one embodiment, SBP rods areinjectable with a 21-gauge needle. In one embodiment, SBP rods areinjectable with a 22-gauge needle. Some rods may be formed forsubcutaneous delivery. Some rods may be formed for other deliveryformats, which may include, but are not limited to, intravitreal,intratympanic, and intraarticular delivery.

Definitions

Absolute value: As used herein, the term “absolute value” describes themagnitude of a numerical number or measurement. The magnitude is listedas a non-negative number, but it can represent both positive andnegative values.

Active pharmaceutical agent (API): As used herein, the term “activepharmaceutical agent,” or “API,” describes the component of apharmaceutical composition that exhibits biological activity.

Cumulative release percentage: As used herein, the term “cumulativerelease percentage” describes the total percentage of a factor releasedfrom a source or depot over the course of a release period. Thispercentage may be determined from the total mass of released factordivided by initial mass of the factor in the source or depot. The “dailyrelease percentage” describes the cumulative release percentage offactor per day. This value may be calculated from the best fit lineslope of a plot of cumulative release percentage overtime.

Effective concentration: As used herein, the tem “effectiveconcentration” refers to the concentration of a compound or factorrequired to elicit a particular response. The concentration needed toelicit half of a complete response is referred to as the “half maximaleffective concentration” or “EC₅₀.” The concentration of compound neededto elicit 80% of a complete response is referred to as the “EC₈₀”. Wherethe compound or factor is inhibitory, the concentration needed to reduceor inhibit the response by half is referred to herein as the halfmaximal inhibitory concentration, or “IC₅₀.”

Initial burst: As used herein, the term “initial burst” refers to a rateof factor release from a source or depot over an initial release period(e.g., after administration or other placement, for example in solutionduring experimental analysis) that is higher than rates during one ormore subsequent release periods.

EQUIVALENTS AND SCOPE

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments in accordance with the invention described herein. The scopeof the present invention is not intended to be limited to the aboveDescription, but rather is as set forth in the appended claims.

In the claims, articles such as “a,” “an,” and “the” may mean one ormore than one unless indicated to the contrary or otherwise evident fromthe context. Claims or descriptions that include “or” between one ormore members of a group are considered satisfied if one, more than one,or all of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention includes embodiments in which more than one, or the entiregroup members are present in, employed in, or otherwise relevant to agiven product or process.

It is also noted that the term “comprising” is intended to be open andpermits but does not require the inclusion of additional elements orsteps. When the term “comprising” is used herein, the term “consistingof” is thus also encompassed and disclosed.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and understanding of one of ordinary skill in the art, valuesthat are expressed as ranges can assume any specific value or subrangewithin the stated ranges in different embodiments of the invention, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise.

In addition, it is to be understood that any particular embodiment ofthe present invention that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Since such embodiments aredeemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the compositions of the invention (e.g., anyantibiotic, therapeutic or active ingredient; any method of production;any method of use; etc.) can be excluded from any one or more claims,for any reason, whether or not related to the existence of prior art.

It is to be understood that the words which have been used are words ofdescription rather than limitation, and that changes may be made withinthe purview of the appended claims without departing from the true scopeand spirit of the invention in its broader aspects.

While the present invention has been described at some length and withsome particularity with respect to the several described embodiments, itis not intended that it should be limited to any such particulars orembodiments or any particular embodiment, but it is to be construed withreferences to the appended claims so as to provide the broadest possibleinterpretation of such claims in view of the prior art and, therefore,to effectively encompass the intended scope of the invention. Thepresent invention is further illustrated by the following nonlimitingexamples.

EXAMPLES Example 1. Formulation of Blank Silk Fibroin Rods Silk FibroinIsolation

Silk yarn, purchased from Jiangsu SOHO International Group, was degummedto remove sericin. 30 grams of cut silk yarn were boiled at 100° C. in 3L of deionized (DI) water with 0.02 M sodium carbonate for 240 minuteswith stirring. The yam was then transferred to a new boiling 0.02 Msodium carbonate aqueous solution and boiled at 100° C. for anadditional 240 minutes with stirring. The total boiling time wasdiscussed in terms of minute boil, or “mb.” The fibroin was then placedin DI water at 60-70° C. for 20 minutes with stirring, and then rinsedwith clean DI water. This process was repeated 3 times. The fibroin wasplaced in clean DI water, stirred for 20 minutes, then rinsed with cleanDI water, and this process was repeated for a total of 3×20 min.-rinsecycles. The fibroin was dried overnight, weighed, and dissolved at 20%(w/v) in a 9.3 M aqueous solution of lithium bromide (fromSigma-Aldrich, St. Louis, Mo.) for 5 hours at 60° C. The resultingfibroin solution was dialyzed against water at 4° C. in a 50 kDaregenerated cellulose dialysis tubing for 48 hours, with 6 water changesto remove the excess salt. The conductivity was recorded after eachwater change with a digital quality tester. When the conductivity wasunder 5 ppm, the fibroin solution was determined to be ready.

The resulting solution was centrifuged for 20 minutes at 3,900 RPM and4° C. to remove insoluble particles. The supernatant was collected, andsamples of the supernatant were diluted at 1:20 and 1:40 in water.Samples for a standard curve were prepared for an A280 assay by dilutingpre-measured fibroin solutions to 5, 2.5, 1.25, 0.625, 0.3125, and 0mg/mL in water. The silk concentration of the 1:20 and 1:40 diluted silkfibroin samples was measured against the standard curve by theabsorbance at 280 nm.

The fibroin solutions were diluted to a final concentration of 3% (w/v)in 10 mM phosphate buffer (from Sigma Aldrich Fine Chemicals, St. Louis,Mo.), pH 7.4, and they were filtered through a 0.2 μm filter using avacuum filter unit. 10 mL of each solution was aliquoted into 50 mLconical tubes, snap frozen in liquid nitrogen for 10 minutes,transferred for 20 minutes in −80° C., and lyophilized for 72 hours.

Formulation of Silk Fibroin Rods

Lyophilized silk fibroin was dissolved in ultrapure water to obtain aconcentration of 40% (w/v). The solution was extruded out of a syringeinto tubing with a variety of diameters, dependent on the indication.For this example, the sample listed in Table 1 was extruded intoapproximately 12 cm lengths of 0.508 mm diameter polyetheretherketone(PEEK) (from Van Waters and Rogers (VWR), PA, USA, product 53500-690).The ends of the tubing were covered in parafilm, and the tubing was thenincubated at 37° C. for 24 hours, after which it was cut to thenecessary size, typically 2 cm lengths, frozen to −80° C. for at leastfour hours, and lyophilized. The final rods contained trace amounts ofpotassium phosphate buffer (with potassium phosphate dibasic andpotassium phosphate monobasic). The final concentration of phosphatebuffer was 133.3 mM.

TABLE 1 Samples of silk-fibroin rods Silk Prep Silk-Fibroin Sample BoilTime Final % No. (min) (w/w) 1 480 100

The resulting rods were imaged via scanning electron microscopy (SEM).The rods were approximately 400 μm in diameter. The outer surfaces andcross-sectional surfaces of the silk-fibroin rods were smooth, with fewto no ridges. The silk-fibroin rods were densely-packed, and thecross-sectional surfaces appeared smooth and contained few to nointernal pores.

Example 2. In Vitro Release of Small Molecules from 1 mm Silk FibroinRods

The silk yarn was purchased from Jiangsu SOHO International Group(Jiangsu, China). Lithium bromide was purchased from Sigma Aldrich (St.Louis, Mo.). The potassium phosphate monobasic and potassium phosphatedibasic were purchased from Sigma Aldrich Fine Chemicals (SAFC) (St.Louis, Mo.). The sodium carbonate and the sodium azide were purchasedfrom Fisher Chemical (Waltham, Mass.). The celecoxib (CXB) was purchasedfrom Cipla (Miami, Fla.).

Silk Fibroin Isolation

Silk yarn, purchased from Jiangsu SOHO International Group, was degummedto remove sericin. 30 grams of cut silk yam were boiled at 100° C. in 3L of deionized (DI) water with 0.02 M sodium carbonate for 240 minuteswith stirring. The yam was then transferred to a boiling 0.02 M sodiumcarbonate aqueous solution and boiled at 100° C. for an additional 240minutes with stirring. The fibroin was then placed in DI water at 60-70°C. for 20 minutes with stirring, and then rinsed with clean DI water.This process was repeated 3 times. The fibroin was placed in clean DIwater, stirred for 20 minutes, then rinsed with clean DI water. Thisprocess was repeated for a total of three 20 minute rinse cycles. Thefibroin was dried overnight, weighed, and dissolved at 20% (w/v) in a9.3 M aqueous solution of lithium bromide (from Sigma Aldrich, St.Louis, Mo.) for 5 hours at 60° C. The resulting fibroin solution wasdialyzed against water at 4° C. in a 50 kDa regenerated cellulosedialysis tubing for 48 hours, with 6 water changes to remove the excesssalt. The conductivity was recorded after each water change with adigital quality tester. When the conductivity was under 5 ppm, thefibroin solution was determined to be ready.

The resulting solution was centrifuged for 20 minutes at 3,900 RPM and4° C. to remove insoluble particles. The supernatant was collected, andsamples of the supernatant were diluted at 1:20 and 1:40 in water.Samples for a standard curve were prepared for an A280 assay by dilutingpre-measured fibroin solutions to 5, 2.5, 1.25, 0.625, 0.3125, and 0mg/mL in water. The silk concentration of the 1:20 and 1:40 diluted silkfibroin samples was measured against the standard curve by theabsorbance at 280 nm.

The silk fibroin solutions were diluted to a final concentration of 3%(w/v) in 10 mM phosphate buffer (from Sigma Aldrich Fine Chemicals, St.Louis Mo.), pH 7.4, and they were filtered through a 0.2 μm filter usinga vacuum filter unit. 10 mL of each solution was aliquoted into 50 mLconical tubes, snap frozen in liquid nitrogen for 10 minutes,transferred for 20 minutes in −80° C., and lyophilized for 72 hours.

1 mm Silk Fibroin Rod Preparation

Lyophilized silk fibroin was dissolved with ultrapure water to obtainsilk concentrations of 20, 30, and 40% (w/v). The relevant amount ofcelecoxib (CXB) (from Cipla. Miami Fla.) was weighed into a 4 mL glassvial. 250 μL of the relevant silk-fibroin solution (for example, Samples8-58-1 through 8-58-3 use 250 μL of 20% (w/v) silk-fibroin to reach 50mg) were then added to the dry CXB. The vial was briefly vortexed. Ametal spatula was then used to manually mix the suspension until itbecame homogeneous. Using the spatula, the viscous suspension was loadedinto the back of a 1 cc. syringe. The viscous mixture was then extrudedout of the syringe into tubing with a variety of diameters, dependent onthe indication. For this example, the samples were extruded intoapproximately 12 cm lengths of 1 mm diameter of either silicon(Grainger, Ill., USA, product number 2VLW4) or polytetrafluoroethylene(PTFE) tubing (from Van Waters and Rogers (VWR), PA, USA) The tubing wassealed with parafilm on both ends and left at 37° C. overnight to inducegelation. The tubing was then cut to the necessary size, typically 2 cmlengths. When the mixture was extruded from the tubing, the rods werefound to hold their shape. The mixture was then frozen at −80° C. for atleast four hours, either within or outside of the tubing. The resultingrods were then lyophilized for approximately 24 hours. Rods were removedfrom the tubing after lyophilization.

The rods are described in Table 2, alongside the concentration of silksolution used in their formulation, the total mass of silk fibroin usedto formulate the rods, the total mass of CXB used to formulate the rods,and the theoretical loading percentages of the silk-fibroin and CXB ineach sample. The term theoretical loading percentage refers to theassumed percentage of a component incorporated in a substance orproduct. The product may be an SBP. The component may be silk fibroin orCXB. The theoretical loading percentage may be in terms of either w/wpercentage, w/v percentage, or v/v percentage. The samples were named bythe process used to prepare and formulate each silk rod. For example,the sample named “480 mb; mm; 20% st; 50 mgsf 150 mgcxb; lyo; 25% sf;75% cxb;” refers to a silk fibroin rod prepared from silk degummed witha 480-minute boil, an extrusion with a 1 mm diameter, a preparation froma 20% stock solution of silk fibroin, a preparation from 50 mg of silkfibroin, a preparation from 150 mg of celecoxib, lyophilization, atheoretical w/w percentage of 25% silk fibroin, and a theoretical w/wpercentage of 75% celecoxib. The final rods contained trace amounts ofpotassium phosphate buffer (with potassium phosphate dibasic andpotassium phosphate monobasic). The final concentration of phosphatebuffer could be converted to (w/w) percentage by multiplying theconcentration (in mM) by 0.0167.

TABLE 2 Theoretical silk fibroin and celecoxib percentages for 1 mm silkrod samples Formulation Silk- Silk- Phosphate Stock Silk Fibroin CXBFibroin CXB Buffer Sample Concentration Mass Mass Final % Final %Concentration No. Sample Name (w/v %) (mg) (mg) (w/w) (w/w) (mM) 8-58-1480 mb; 1 mm; 20% st; 20 50 150 25 75 41.7 50 mgsf; 150 mgcxb; lyo; 25%sf; 75% cxb 8-58-2 480 mb; 1 mm; 20% st; 20 50 200 20 80 37 50 mgsf; 200mgcxb; lyo; 20% sf; 80% cxb — 480 mb; 1 mm; 20% st; 20 50 250 16.7 83.333.3 50 mgsf; 250 mgcxb; lyo; 16.7% sf; 83.3% cxb 8-58-4 480 mb; 1 mm;30% st; 30 75 150 33.3 66.7 62.5 75 mgsf; 150 mgcxb; lyo; 33.3% sf;66.7% cxb 8-58-5 480 mb; 1 mm; 30% st; 30 75 200 27.3 72.2 55.6 75 mgsf;200 mgcxb; lyo; 27.3% sf; 72.2% cxb 8-58-6 480 mb; 1 mm; 30% st; 30 75250 23 77 50 75 mgsf; 250 mgcxb; lyo; 23% sf; 77% cxb 8-58-7 480 mb; 1mm; 40% st; 40 100 150 40 60 83.3 100 mgsf; 150 mgcxb; lyo; 40% sf; 60%cxb 8-58-8 480 mb; 1 mm; 40% st; 40 100 200 33.3 66.7 74.1 100 mgsf; 200mgcxb; lyo; 33.3% sf; 66.7% cxb 8-58-9 480 mb; 1 mm; 40% st; 40 100 25028.6 71.4 66.7 100 mgsf; 250 mgcxb; lyo; 28.6% sf; 71.4% cxb

The resulting silk fibroin rods were imaged via scanning electronmicroscopy (SEM), seen in FIG. 1A, FIG. 1B, FIG. 1C, 1D. The rods wereapproximately 1000 μm in diameter. The silk-fibroin-CXB-rods weredensely packed. The outer surfaces and cross-sectional surfaces of thesilk-fibroin-CXB rods had ridges that appeared approximately 15 μm inlength. The cross-sectional images of the silk-fibroin-CXB rodscontained pores ranging in size from approximately 10-75 μm in length.

In Vitro Release Experiments

The diameter of the silk-fibroin rods was measured using digitalcalipers. The rods were cut to 1 cm lengths to standardize release, andthe weights of the rods were recorded. The density of the rods wascalculated for each preparation. The rods from the tubing were placedinto 45 mL of phosphate buffer (from Sigma Aldrich Fine Chemicals, St.Louis, Mo.), pH 7.4, 2% (v/v)Polysorbate-80 (from Croda, Snaith UK.),and 0.05% (w/v) sodium azide (from Fisher Chemical, Waltham Mass.). Thisbuffer ensured that the release was conducted under sink conditions (≥5×saturated solubility). The samples were incubated at 37° C. with gentleshaking. 1 mL of the release medium was taken at each timepoint(typically 1, 4, 7, 10, and 14 days and then weekly thereafter) andreplaced with fresh media. The release medium was then analyzed viaultra-performance liquid chromatography (UPLC) to determine CXBconcentration.

The silk fibroin rods demonstrated near zero-order kinetics for CXBrelease, with a low initial burst of 5-20%. The release rates of CXBwere tuned by altering the density, CXB loading, and silk fibroinconcentration. The CXB was released over the course of 1-3 months.

Example 3. In Vitro Release of Small Molecules from 0.5 mm Silk FibroinRods

The silk yarn was purchased from Jiangsu SOHO International Group(Jiangsu, China). Lithium bromide was purchased from Sigma Aldrich (St.Louis, Mo.). The potassium phosphate monobasic and potassium phosphatedibasic were purchased from Sigma Aldrich Fine Chemicals (SAFC) (St.Louis, Mo.). The sodium carbonate and the sodium azide were purchasedfrom Fisher Chemical (Waltham, Mass.). The celecoxib (CXB) was purchasedfrom Cipla (Miami, Fla.).

0.5 mm Silk Fibroin Rod Preparation

Silk-fibroin (from Jiangsu SOHO International Corporation) was isolatedas described in the preparation of the silk fibroin rods with noadditives. Briefly, silk yam, purchased from Jiangsu SOHO InternationalGroup, was degummed to remove sericin. 30 grams of cut silk yam wereboiled at 100° C. in 3 L of deionized (DI) water with 0.02 M sodiumcarbonate with stirring. The yam was then transferred to a new boiling0.02 M sodium carbonate aqueous solution and boiled at 100° C. foradditional time with stirring. The total boiling time was discussed interms of minute boil, or “mb.” The silk fibroin was boiled for either atotal time of 480 or 120 minutes while being degummed. The total boilingtime was discussed in terms of minute boil, or “mb.” Longer boilingtimes produced silk fibroin with lower average molecular weights ofapproximately 5-60 kDa.

The fibroin was then placed in DI water at 60-70° C. for 20 minutes withstirring, and then rinsed with clean DI water. This process was repeated3 times. The fibroin was placed in clean DI water, stirred for 20minutes, then rinsed with clean DI water, and this process was repeatedfor a total of 3×20 min.-rinse cycles. The fibroin was dried overnight,weighed, and dissolved at 20% (w/v) in a 9.3 M aqueous solution oflithium bromide (from Sigma-Aldrich, St. Louis, Mo.) for 5 hours at 60°C. The resulting fibroin solution was dialyzed against water at 4° C. ina 50 kDa regenerated cellulose dialysis tubing for 48 hours, with 6water changes to remove the excess salt. The conductivity was recordedafter each water change with a digital quality tester. When theconductivity was under 5 ppm, the fibroin solution was determined to beready. The silk fibroin solution was centrifuged for 20 minutes at 3.900RPM and 4° C. to remove insoluble particles. Solutions were diluted to afinal concentration of 3% (w/v) in 10 mM phosphate buffer, pH 7.4,filtered through a 0.22 μm filter, frozen in liquid nitrogen, andlyophilized for 72 hours.

Lyophilized silk-fibroin was dissolved with ultrapure water to obtainconcentrations of 20, 30, and 40% (w/v). The relevant amount of CXB(from Cipla, Miami Fla.) was weighed into a 4 mL glass vial. 250 μL ofthe relevant silk-fibroin solution was then added to the dry CXB, andthe vial was then briefly vortexed. A metal spatula was used to manuallymix the suspension until it was homogeneous. Using the spatula, theviscous suspension was loaded into the back of a 1 cc. syringe. Theviscous mixture was extruded out of the syringe into tubing with avariety of diameters, dependent on the indication. For this example, thesamples listed in Table 3 were extruded into approximately 12 cm lengthsof 0.508 mm diameter PEEK tubing (from Van Waters and Rogers (VWR), PA,USA, product 53500-690). The tubing was then sealed on both ends withparafilm and left at 37° C. for 24 hours or overnight for gelation. Thetubing was cut to the necessary size, typically 2 cm lengths. Half ofthe samples were frozen to −80° C. for at least four hours andlyophilized, while half of the samples were oven dried at 60° C. for 16hrs. The samples were named by the process used to prepare and formulateeach silk rod. For example, the sample named “480 mb; 0.5 mm; 40% st;100 mgsf; 200 mgcxb; lyo; 33.3% sf; 66.7% cxb” refers to a silk fibroinrod prepared from silk degummed with a 480-minute boil, an extrusionwith a 0.5 mm diameter, a preparation from a 40% stock solution of silkfibroin, a preparation from 100 mg of silk fibroin, a preparation from200 mg of celecoxib, lyophilization, a theoretical w/w percentage of33.3% silk fibroin, and a theoretical w/w percentage of 66.7% celecoxib.The final rods contained trace amounts of potassium phosphate buffer(with potassium phosphate dibasic and potassium phosphate monobasic).The final concentration of phosphate buffer could be converted to (w/w)percentage by multiplying the concentration (in mM) by 0.0167.

TABLE 3 Theoretical silk fibroin and celecoxib percentages for 0.5 mmsilk rod samples Stock Silk Concentration Prep Silk- Silk- Phosphate ofSilk for Boil Fibroin CXB Fibroin CXB Buffer Sample Sample FormulationTime Mass Mass Final % Final % Conc. No. Name (w/v %) (min) (mg) (mg)(w/w) (w/w) (mM) — — 480 50 200 20 80 37 — — 480 75 200 27.3 72.2 55.6 —— 480 100 100 50 50 95.2 — — 480 100 150 40 60 83.3 8-65-6 480 mb; 40480 100 200 33.3 66.7 74.1 0.5 mm; 40% st; 100 mgsf; 200 mgcxb; lyo;33.3% sf; 66.7% cxb — — 480 100 250 28.6 71.4 66.7 — — 120 50 200 20 8037 — — 120 75 200 27.3 72.2 55.6 — — 120 100 150 40 60 83.3

The resulting lyophilized rods were photographed (see FIG. 2A) of imagedvia SEM (see FIG. 2B, FIG. 2C, and FIG. 2D). The rods were approximately400 μm in diameter, and the rod in FIG. 2A, FIG. 2B, FIG. 2C, and FIG.2D had a diameter of 430 μm. The silk-fibroin rods were densely packedwith an even distribution of the API. The outer surfaces andcross-sectional surfaces of the silk-fibroin rods loaded with CXB hadridges that appeared approximately 15 μm in length. Furthermore, thecross-sectional images of the silk-fibroin rods with celecoxib containedfew small pores.

In Vitro Release Experiments

The rods were cut to 1 cm lengths to standardize release, and theweights of the rods were recorded. The densities of the rods werecalculated for each preparation. The rods were placed into 45 mL ofphosphate buffer, pH 7.4, 0.3% (v/v) Polysorbate-80 (from Croda, SnaithUK), and 0.05% (w/v) sodium azide (from Fisher Chemical, Waltham Mass.).This buffer ensured that the release was conducted under sink conditions(≥5× saturated solubility). A suspension of CXB containing 800 μg CXBwas used as a control. The samples were incubated at 37° C. with gentleshaking. 1 mL of the release medium was taken at each timepoint(typically 1, 4, 7, 10, and 14 days and then weekly thereafter) andreplaced with fresh media. The release medium was then analyzed via UPLCat 260 nm to determine CXB concentration.

The silk fibroin rods demonstrated near zero-order kinetics for CXBrelease, with a low initial burst of 15%. The release rates of CXB couldbe modulated by altering the silk molecular weight, CXB loading, and themethod of drying the silk fibroin rods. The CXB was released over thecourse of 1-3 months. The rods with the 0.5 mm diameter displayed afaster release, when compared to the 1 mm rods, due to the largersurface area to volume ratio of the smaller rods.

Example 4. In Vitro Release of Small Molecules from Silk Fibroin Gels

All formulations were prepared with silk yam purchased from SOHO. Thesilk hydrogels were prepared with celecoxib (CXB) (from Cipla, MiamiFla.). The poloxamer-188 (P188), sodium chloride, and hydrochloric acidwere from Sigma-Aldrich (St. Louis, Mo.), while the PEG4 kDa was fromClariant, Charlotte N.C. Polysorbate-80 was purchased from Croda (SnaithUK). Potassium phosphate monobasic and potassium phosphate dibasic werepurchased from Sigma Aldrich Fine Chemical (SAFC, St. Louis Mo.).Phosphate buffered saline was purchased from Gibco (USA).

Formulation of Silk Fibroin Hydrogels

Silk fibroin hydrogels were formulated with poloxamer-188 (P188) (fromSigma. St. Louis, Mo.) or polyethylene glycol 4000 Da (PEG 4k) (fromClariant, Charlotte N.C.). These hydrogels were formulated withcelecoxib, the delivery of which was monitored. To prepare theformulations, a 27.8% suspension of celecoxib (CXB) in 0.79% polysorbate80 as well as a stock solution of phosphate buffer (315 mM, pH=7.4) wasused to dissolve either 120 mb or 480 mb silk fibroin and added to asyringe. Excipient solutions were then prepared with varyingcombinations of sodium chloride, PEG4 kDa. P188, and/or hydrochloricacid and added to a second syringe. Excipient solutions were prepared sothat a 0.75:1 mix of silk-fibroin solution:excipient solution wouldresult in the desired final formulations, with an osmolarity of 280mOsm. The two syringes were then connected via a B Braun fluiddispensing connector, and the contents of the two syringes were mixedback and forth until homogeneous (at least 25 times). The syringes werethen capped with a sterile syringe cap and incubated on a rotator at 37°C. for 24 hours. Syringes were stored at 4° C. until analysis.

Formulations were prepared as described in Table 4A and Table 4B, witheither high molecular weight (HMW or 120 mb, with an average molecularweight of 100-300 kDa) or low molecular weight (LMW or 480 mb, with anaverage molecular weight of about 30-60 kDa) silk fibroin. Longerboiling times, measured in “minute boil” or “mb”, produced silk fibroinwith smaller molecular weights. The samples in Table 4A and Table 4B arenamed by the process used to prepare and formulate each hydrogel. Forexample, in the sample named 120 mb; hyd; 27.8% cxbst; 5% SFf; 10% CXBf;40% PEG4kf, “120 mb” refers to silk degummed with a 120-minute boil,“hyd” refers to the formulation of the sample as a hydrogel, “27.8%cxbst” refers to a preparation from a stock solution of 27.8% ofcelecoxib, “5% SFf” refers to a formulation with 5% (w/v) silk fibroin.“10% CXBf” refers to a formulation with 10% (w/v) celecoxib, and “40%PEG4kf” refers to a formulation with 40% PEG 4 kDa. Some hydrogels wereprepared with P188 (% P188f). The hydrogels were injectable through a27-gauge, ½ inch needle. The hydrogels were formulated with varying silkfibroin molecular weights, gelling excipients, and silk fibroinconcentrations. The hydrogels were formulated under aqueous conditions,with tight control of osmolarity and pH. The pH was measured with aB30PCI Benchtop Multi Parameter Meter—PH, Conductivity, ISE (VWR Catalog#89231-696), with a glass probe (VWR Catalog #89231-592). All hydrogelshad a final phosphate buffer concentration of 22 mM.

TABLE 4A Descriptions of hydrogels prepared loaded with celecoxib Silk-Min. fibroin Excipient CXB NaCl HCl Sample Sample Boil Conc. conc. Conc.Conc. Conc. No. name Description (mb) % Excipient % % (mg/mL) (mM) 168-1120 mb; hyd; 5% 120 mb 120 5 PEG 4k 40 10 2.95 15 27.8% cxbst; with PEG5% SFf; 4k 10% CXBf; 40% PEG4kf 168-2 120 mb; hyd; 3% 120 mb 120 3 PEG4k 40 10 2.95 15 27.8% cxbst; with PEG 3% SFf; 4k 10% CXBf; 40% PEG4kf168-3 120 mb; hyd; 5% 120 mb 120 5 P188 10 10 5.97 0 27.8% cxbst; withP188 5% SFf; 10% CXBf; 10% P188f 168-4 120 mb; hyd; 3% 120 mb 120 3 P18810 10 5.99 0 27.8% cxbst; with P188 3% SFf; 10% CXBf; 10% P188f 168-5480 mb; hyd; 5% 480 mb 480 5 PEG 4k 40 10 2.87 15 27.8% cxbst; with PEG5% SFf; 4k 10% CXBf; 40% PEG4kf 168-6 480 mb; hyd; 3% 480 mb 480 3 PEG4k 40 10 2.91 15 27.8% cxbst; with PEG 3% SFf; 4k 10% CXBf; 40% PEG4kf168-7 480 mb; hyd; 5% 480 mb 480 5 P188 10 10 5.90 0 27.8% cxbst; withP188 5% SFf; 10% CXBf; 10% P188f 168-8 480 mb; hyd; 3% 480 mb 480 3 P18810 10 5.94 0 27.8% cxbst; with P188 3% SFf; 10% CXBf; 10% P188f 168-9480 mb; hyd; 2% 480 mb 480 2 P188 10 10 5.96 0 27.8% cxbst; with P188 2%SFf; 10% CXBf; 10% P188f

TABLE 4B Properties of the hydrogels prepared loaded with celecoxibStandard Actual Deviation Sample CXB % of Actual Mass No. Sample name(w/v) CXB % pH Replicate (mg) 168-1 120 mb; hyd; 9.5 0.8 6.78 A 49.7827.8% cxbst; 5% SFf; B 54.35 10% CXBf; C 53.45 40% PEG4kf 168-2 120 mb;hyd; 9.5 0.3 6.82 A 52.89 27.8% cxbst; 3% SFf; B 54.44 10% CXBf; C 50.4840% PEG4kf 168-3 120 mb; hyd; 11.9 3.5 7.1 A 56.07 27.8% cxbst; 5% SFf;B 53.96 10% CXBf; 10% P188f C 49.44 168-4 120 mb; hyd; 9.6 0.8 7.06 A50.42 27.8% cxbst; 3% SFf; B 54.12 10% CXBf; 10% P188f C 50.14 168-5 480mb; hyd; 9.3 0 7.15 A 51.75 27.8% cxbst; 5% SFf; B 49.55 10% CXBf; C55.33 40% PEG4kf 168-6 480 mb; hyd; 9.2 0.7 6.98 A 56.38 27.8% cxbst; 3%SFf; B 50.92 10% CXBf; C 49.08 40% PEG4kf 168-7 480 mb; hyd; 8.7 0.17.16 A 55.12 27.8% cxbst; 5% SFf; B 51.59 10% CXBf; 10% P188f C 54.18168-8 480 mb; hyd; 9.8 0.6 7.15 A 55.9 27.8% cxbst; 3% SFf; B 53.53 10%CXBf; 10% P188f C 56.17 168-9 480 mb; hyd; 9.4 1.6 7.13 A 52.39 27.8%cxbst; 2% SFf; B 54.56 10% CXBf; 10% P188f C 53.38

In Vitro Release Experiments

In triplicate, 50 mg of each formulation was weighed into half of a #4gelatin capsule (MyHerbar, Dallas Tex.). It had previously been shownthat the solubility of celecoxib in this release media was 850 μg/mL. 45mL of this release media allowed for 38 mg CXB solubility. This mediaensured sink conditions (greater than or equal to 5 times the CXBsolubility) were maintained throughout the course of the study. Thetubes were capped and incubated at 37° C. with shaking. 1 mL of therelease media was collected from each sample at each timepoint andreplaced with 1 mL fresh media. At each timepoint, the tubes were leftto stand on end for at least 30 minutes to allow the formulation tosettle prior to taking the sample. Release media was analyzed by HPLC-UV(Agilent 1290 HPLC system) at 260 nm. Controls were prepared at Day 0 byweighing 50 mg of each formulation in triplicate in separate 20 mL glassvials. Methanol was added to each sample to extract CXB. Samples wereplaced on a shaker at room temperature for 24 hours. The supernatant wasanalyzed by HPLC-UV to determine CXB loading. The results of the invitro release experiments, seen in Table 5A, and Table 5B wereconsistent with first-order kinetics, with initial bursts from 25%-100%.All tested hydrogel formulations released the small molecule up to onemonth after the start of the experiment.

TABLE 5A In vitro release kinetics for hydrogels loaded with celecoxib;average cumulative percentage of API released Time Sample No. (days)168-1 168-2 168-3 168-4 168-5 168-6 168-7 168-8 168-9 0 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 1 24.59 42.53 51.15 42.75 30.64 96.2443.26 27.68 46.90 4 50.91 67.96 73.54 69.47 56.39 96.99 73.38 51.5674.89 7 67.59 81.51 79.05 82.51 75.78 96.32 87.00 66.01 87.51 14 79.4386.60 75.51 88.35 86.61 94.02 94.64 80.88 93.81 25 96.86 98.22 85.72104.14 102.82 105.89 110.51 100.24 105.61 29 95.90 96.43 82.39 99.86102.21 100.32 105.56 95.38 102.51

TABLE 5B Standard deviations of the average cumulative percentage of APIreleased from the in vitro release kinetics experiments for hydrogelsloaded with celecoxib Time Sample No. (days) 168-1 168-2 168-3 168-4168-5 168-6 168-7 168-8 168-9 0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.000.00 1 3.04 7.33 2.96 13.35 3.38 1.36 15.21 5.30 10.79 4 5.07 5.88 1.7711.33 5.30 2.46 19.62 6.69 9.42 7 5.21 4.57 4.00 9.15 10.63 2.75 16.716.27 8.46 14 4.56 2.18 7.29 4.63 5.20 3.93 11.75 4.33 1.02 25 2.68 4.979.73 8.47 4.63 0.74 6.66 4.63 3.76 29 1.80 3.88 8.30 4.87 3.02 2.68 7.772.59 2.74

For the hydrogels prepared with P188, the initial burst was the highestfor the hydrogel with 5% (w/v) high molecular weight silk fibroin, asseen in Table 5A. The hydrogel with 3% (w/v) low molecular weight silkfibroin had the lowest initial burst of therapeutic agent. The remaininghydrogels had initial bursts of a similar magnitude, the values of whichwere between those of the 5% (w/v) high molecular weight and the 3^(%)(w/v) low molecular weight silk fibroin hydrogels. The hydrogels (withP188) with higher concentrations of silk fibroin demonstrated greaterinitial bursts of API in comparison with the corresponding hydrogelswith lower concentrations of silk fibroin. In addition, the hydrogels(with P188) prepared from higher molecular weight silk fibroin alsodemonstrated greater initial bursts of API than the correspondinghydrogels with lower molecular weight silk fibroin.

For the hydrogels prepared with PEG4k, the initial burst was the highestfor the hydrogel prepared with 3% (w/v) low molecular weight silkfibroin, followed by the hydrogel prepared with 3% (w/v) high molecularweight silk fibroin. The hydrogel prepared the with 5% (w/v) highmolecular weight silk fibroin had the lowest initial burst, as seen inTable 5A. The hydrogels (with PEG4k) prepared from higher molecularweight silk fibroin demonstrated lower initial bursts of API than thehydrogels prepared from lower molecular weight silk fibroin. Inaddition, the hydrogel (with PEG4k) with a lower concentration of silkfibroin demonstrated a greater initial burst of API than thecorresponding hydrogel with a higher concentration of silk fibroin.

The use of excipients with different molecular weights also revealed apattern in the initial burst of therapeutic agent from the hydrogels.While both hydrogels were prepared at the same osmolarity, excipientsused had different molecular weights. PEG4k had a molecular weight of 4kDa, while P188 had a molecular weight of 8.4 kDa. The molecular weightof the excipient modulated the observed trends in the initial burstpercentages. Hydrogels prepared from excipients with higher molecularweights demonstrated a direct relationship between the concentration ofsilk fibroin and the initial burst and a direct relationship between themolecular weight of the silk fibroin and the initial burst. Meanwhile,hydrogels prepared from excipients with lower molecular weightsdemonstrated an inverse relationship between the concentration of silkfibroin and the initial burst and an inverse relationship between themolecular weight of the silk fibroin and the initial burst.

Example 5. Biocompatibility of Silk Fibroin Rods and Hydrogels

Silk fibroin rods or silk fibroin hydrogels were formulated with ageneric NSAID. The silk fibroin rods had a diameter of 430 μm and alength of 10 mm. Silk fibroin hydrogels were formulated with and without100 mg/mL NSAID. The rods or hydrogels were administered to healthyrabbits as 100 μL injections in a 27-gauge needle. The rods werepre-loaded into sterile 21G, 1″ needles with pieces of 28G wire werepre-cut, sterilized and placed into the needle from the hub. The needlewas placed (as described below) and the formulation was pushed into theintravitreal space, 2 mm posterior to the limbus using the length of 28Gwire. The wire extended past the end of needle 3-4 mm to ensure fullinjection. A lid speculum was inserted into the rabbit's left eye lid.The conjunctiva was drenched with BSS solution from a sterile dropper(3-5 drops). 1-2 drops of betadine solution was applied allowing 30seconds after administration. One additional drop of betadine solutionwas applied followed by injection of the formulation using adouble-plane tunnel technique (the sclera was penetrated at 15°-30° thenthe needle is repositioned to a 45°-60° angle while the sclera was stillengaged, the formulation was delivered and the needle removed at a 90°angle). Following injection, the central retinal artery was examined viaindirect ophthalmoscopy to confirm perfusion and 1-2 drops of betadinesolution were added to the conjunctiva prior to removal of the speculum.The silk fibroin compositions remained cohesive or in one piece in theintravitreal space. The subjects experienced normal intraocularpressures, no local inflammation, no hemorrhage, and no othercomplications. The silk fibroin rods and hydrogels were tolerated in theintravitreal space.

Example 6. Tolerability Studies

The tolerability of silk fibroin solutions, hydrogels, and rods wasmonitored in rabbits, rats, and dogs. All materials studied werewell-tolerated clinically. The hydrogel material was observed tointegrate into tissue with minimal inflammation, which was consistentwith a transient local foreign body reaction. No adverse reactions werenoted.

Example 7. Human Whole Blood Assay

Whole human blood was exposed to soluble silk fibroin for 24 hours at37° C. and assessed for inflammation. Lipopolysaccharide (LPS) was usedas a positive stimulator of the inflammatory marker TNF-α, in wholeblood. The experiments were conducted in the presence and absence of LPSto determine whether any formulation constituent had the activity ofpotentiating a known inflammatory signal. Plasma was collected at theend of the experiment and analyzed by enzyme-linked immunosorbent assay(ELISA) for TNF-α (FIG. 3). The experiments were performed with bloodfrom 5 donors (FIG. 3) and repeated with 2 additional donors. The silkfibroin did not increase the release of TNF-α, and other inflammatorymarkers such as PGE2. The results were consistent with multiple silkfibroin formats, such as silk fibroin with different molecular weights,hydrogels, 3D fibroin scaffolds, and hydrogel extracts. No signs oflocal sensitization were detected after extended exposure.

Example 8. Measurements of Diameter, Density and In Vitro Experiments on1 mm Celecoxib Loaded Silk Fibroin Rods

The diameter of the silk-fibroin rods was measured using digitalcalipers. The rods were cut to 1 cm lengths to standardize release, andthe weights of the rods were recorded. The density of the rods wascalculated for each formulation. As seen in Table 6, the experimentaldata revealed that the samples generated at each theoretical w/w %formed silk rods with a diameter slightly below 1 mm, the theoreticalsilk rod diameter. In addition, most of the samples yielded silk rodswith a density near 1 g/mL. In Table 6, “Std. Dev.” Refers to standarddeviation.

TABLE 6 Observed diameter and density of 1 mm silk-fibroin rods DensitySample Diameter Density Std. Dev. No. Sample Name (mm) (g/mL) (g/mL)8-58-1 480 mb; 1 mm; 20% st; 50 mgsf; 150 mgcxb; lyo; 0.93 0.79 0.05 25%sf; 75% cxb 8-58-2 480 mb; 1 mm; 20% st; 50 mgsf; 200 mgcxb; lyo; 0.950.83 0.08 20% sf; 80% cxb 8-58-4 480 mb; 1 mm; 30% st; 75 mgsf; 150mgcxb; lyo; 0.88 1.00 0.06 33.3% sf; 66.7% cxb 8-58-5 480 mb; 1 mm; 30%st; 75 mgsf; 200 mgcxb; lyo; 0.92 1.09 0.14 27.3% sf; 72.2% cxb 8-58-6480 mb; 1 mm; 30% st; 75 mgsf; 250 mgcxb; lyo; 0.96 1.07 0.05 23% sf;77% cxb 8-58-7 480 mb; 1 mm; 40% st; 100 mgsf; 150 mgcxb; lyo; 0.88 1.190.07 40% sf; 60% cxb 8-58-8 480 mb; 1 mm; 40% st; 100 mgsf; 200 mgcxb;lyo; 0.91 1.28 0.05 33.3% sf; 66.7% cxb 8-58-9 480 mb; 1 mm; 40% st; 100mgsf; 250 mgcxb; lyo; 0.92 1.30 0.11 28.6% sf; 71.4% cxb

Extraction controls were run to determine celecoxib (CXB) loading in therods. Pre-weighed, 1 cm lengths of the rods were placed into 5 mL of100% methanol, vortexed, and sonicated. The samples were left to shakeovernight at room temperature. The methanol was then analyzed for CXBloading via UPLC. For most samples, the experimental loading percentageof CXB of the silk rods was lower than the theoretical loadingpercentage of CXB, as seen in Table 7. Many of the samples had actualCXB loadings around 8% lower than the theoretical CXB loading.

TABLE 7 Celecoxib loading after extraction for 1 mm rods TheoreticalActual Standard Sample CXB % CXB % Dev. of Density No. Sample Name (w/w)(w/w) CXB % (g/mL) 8-58-1 480 mb; 1 mm; 20% st; 50 mgsf; 150 mgcxb; 7564.1 4.4 0.79 lyo; 25% sf; 75% cxb 8-58-2 480 mb; 1 mm; 20% st; 50 mgsf;200 mgcxb; 80 70.4 0.8 0.83 lyo; 20% sf; 80% cxb 8-58-4 480 mb; 1 mm;30% st; 75 mgsf; 150 mgcxb; 66.7 59.3 1.1 1.00 lyo; 33.3% sf; 66.7% cxb8-58-5 480 mb; 1 mm; 30% st; 75 mgsf; 200 mgcxb; 72.2 65.6 4.1 1.09 lyo;27.3% sf; 72.2% cxb 8-58-6 480 mb; 1 mm; 30% st; 75 mgsf; 250 mgcxb; 7774.0 9.0 1.07 lyo; 23% sf; 77% cxb 8-58-7 480 mb; 1 mm; 40% st; 100mgsf; 150 mgcxb; 60 59.7 15.7 1.19 lyo; 40% sf; 60% cxb 8-58-8 480 mb; 1mm; 40% st; 100 mgsf; 200 mgcxb; 66.7 62.7 2.4 1.28 lyo; 33.3% sf; 66.7%cxb 8-58-9 480 mb; 1 mm; 40% st; 100 mgsf; 250 mgcxb; 71.4 65.9 4.5 1.30lyo; 28.6% sf; 71.4% cxb

For the release experiments, the rods were placed into 45 mL ofphosphate buffer, pH 7.4, 2% (v/v) Polysorbate-80 (from Croda, SnaithUK), and 0.05% (w/v) sodium azide (from Fisher Chemical, Waltham Mass.).This buffer ensured that the release was conducted under sink conditions(≥5× saturated solubility). The samples were incubated at 37° C. withgentle shaking. 1 mL of the release medium was taken at each timepoint(typically 1, 4, and 7 days and then weekly thereafter). The releasemedium was then analyzed via ultra-performance liquid chromatography(UPLC) to determine CXB concentration. The results were shown in Table8A and Table 8B.

TABLE 8A In vitro release kinetics of celecoxib from 1 mm silk- fibroinrods; cumulative percentage of API released Sample No. Day 8-58-1 8-58-28-58-4 8-58-5 8-58-6 8-58-7 8-58-8 8-58-9 0 0 0.0 0.0 0 0.0 0.0 0.0 0.01 18.6 15.0 10.0 9.0 7.1 5.6 6.2 5.7 4 33.3 29.1 21.4 17.7 14.3 11.112.8 11.8 7 48.4 41.5 28.1 25.4 21.2 16.8 18.7 17.6 11 60.3 51.2 37.434.2 27.8 22.2 25.0 23.8 14 66.6 58.8 41.6 37.8 32.1 25.1 28.2 26.5 2181.9 73.7 53.2 50.0 42.0 34.1 36.2 33.8 28 98.0 88.3 65.5 59.0 51.4 42.343.5 42.3 35 96.9 91.4 67.8 62.6 54.4 40.9 48.3 45.0 42 93.7 91.5 66.761.6 54.8 40.9 49.3 44.3 49 101.3 96.3 76.3 71.0 62.8 47.8 54.8 51.2 5698.1 95.4 79.1 73.5 66.8 49.9 58.5 52.5 64 97.2 102.0 84.4 77.6 72.552.9 60.9 57.3 70 — — 88.1 81.2 73.6 57.4 65.5 60.8 76 — — 89.6 83.275.0 58.1 66.9 62.8 84 — — 94.9 87.3 79.3 61.7 71.0 65.8 98 — — 116.8106.1 98.6 75.7 88.8 82.6 112 — — 118.2 108.5 103.9 81.1 96.2 87.7 126 —— 115.1 106.6 103.6 83.4 101.8 91.8 147 — — — — — 92.2 111.6 100.5 162 —— — — — 98.9 121.7 108.8 176 — — — — — 103.1 138.4 114.6 190 — — — — —104.9 124.2 115.4 204 — — — — — 107.1 123.2 116.2

TABLE 8B Standard deviations of the data from the in vitro releasekinetics of celecoxib from 1 mm silk-fibroin rods; cumulative percentageof API released Sample No. Day 8-58-1 8-58-2 8-58-4 8-58-5 8-58-6 8-58-78-58-8 8-58-9 0 0 0.00 0.00 0 0.00 0.00 0.00 0.00 1 3.7 1.94 0.42 0.90.32 0.16 0.05 0.30 4 4.3 2.80 3.95 0.5 0.19 0.24 0.54 0.61 7 5.7 3.311.22 0.5 0.22 0.40 0.32 0.75 11 6.9 2.87 0.58 0.7 0.49 0.80 0.90 0.74 146.3 3.33 0.98 0.7 0.94 0.64 0.77 0.80 21 6.8 4.21 3.11 6.0 2.14 4.440.98 2.17 28 6.3 4.11 0.97 1.2 1.31 2.23 2.76 1.84 35 6.3 2.93 1.40 1.51.98 1.74 3.05 1.79 42 3.46 7.23 2.09 1.6 2.20 1.43 2.84 0.94 49 2.913.96 2.62 1.2 1.93 1.69 1.86 1.61 56 2.18 5.20 2.95 1.0 3.00 2.08 2.121.43 64 5.31 7.87 8.65 1.1 7.93 3.39 1.80 1.47 70 — — 2.85 1.6 2.52 2.283.04 2.64 76 — — 3.28 0.8 1.93 3.36 3.57 3.01 84 — — 3.93 1.9 2.43 3.684.64 3.83 98 — — 6.36 0.8 3.45 4.39 7.17 4.62 112 — — 8.59 1.5 3.88 5.167.89 4.77 126 — — 7.38 1.3 3.87 6.56 11.24 5.32 147 — — — — — 8.93 16.88.89 162 — — — — — 9.82 20.4 7.60 176 — — — — — 10.41 12.5 8.53 190 — —— — — 9.85 16.7 10.83 204 — — — — — 9.42 10.6 9.36

The data demonstrated near-zero-order release kinetics. Each silkfibroin rod sample experienced an initial burst of API release as seenin Table 9 followed by the continued gradual release of the therapeuticagent at a slower rate. The initial burst of API release from the rodsranged from about 5-20% of the API loaded into the rods by mass. Thetheoretical loading percentage of CXB affected the initial burst of APIrelease. Higher percentages of silk fibroin in the theoreticalloading(w/w)percentages of silk fibroin correlated with lower initialburst rates. This inverse relationship between the amount of silkfibroin in the rods and the initial burst rate was evident across allsamples. Sample 8-58-1 reached complete release by day 35, and 8-58-2reached completion by day 64. Samples 8-58-4 and 8-58-5, reachedcomplete release by day 98. Sample 8-58-6 reached complete release byday 112.

TABLE 9 Celecoxib release rates for 1 mm rods Initial Ratio Ratio Dailyburst:Daily Sample CXB:SF CXB:SF Initial Release % release Density No.Theoretical Actual Burst % (at 64 days) (at 64 days) (g/mL) 8-58-1 3.01.8 18.6 1.34 13.9 0.79 8-58-2 4.0 2.4 15.0 1.42 10.6 0.83 8-58-4 2.01.5 10.0 1.19 8.4 1.00 8-58-5 2.6 1.9 9.0 1.11 8.1 1.09 8-58-6 3.3 2.87.1 1.04 6.8 1.07 8-58-7 1.5 1.5 5.6 0.76 7.4 1.19 8-58-8 2.0 1.7 6.20.90 6.9 1.28 8-58-9 2.5 1.9 5.7 0.82 6.9 1.30

The kinetics data demonstrated the possible existence of a relationshipbetween the rate of API release and the (w/w) ratio of API to silkfibroin for the 1 mm silk fibroin rods. These ratios were calculated forboth the theoretical loading and the actual loading of the rods. The useof each formulation in a device or product might depend on the desiredamount of API released in the time frame of interest. For example, if asmaller amount of the API needed to be released in the designated timeframe, the formulations from Samples 8-58-7 through 8-58-9 would be mosteffective. As seen in Table 8A and Table 9, the release duration of CXBwas related to the rod density, with increased density resulting inlonger release times and slower release rates. The rods with a higherdensity also demonstrated a lower daily release percentage and lowerinitial burst percentages. Daily release percentage was defined as theweight percent of the total API released per day, and it was calculatedas the slope of the plot of cumulative release over time. We have shownthe daily release percentages calculated for the first 64 days of thestudy. The rod density was tuned by varying the starting concentrationof the silk-fibroin used during formulation. For example, theformulations prepared with 40% (w/v) silk-fibroin solution had thehighest densities of 1.30, 1.28, and 1.19 g/mL, while the formulationsprepared with 20% (w/v) silk-fibroin had the lowest densities of 0.83and 0.79 g/mL. The initial burst and release rate decreased withincreasing density. Ultimately, the samples with a density below 1.0g/mL reached complete release about 64 days or less, the samples with adensity between 1.0 g/mL and 1.1 g/mL reached complete release in about98 days, and the samples with a density above 1.1 g/mL reached completerelease in greater than 98 days. The higher density rods represented amore tightly packed CXB/fibroin formulation. Since both the CXB as wellas the formulated silk-fibroin were hydrophobic, this lead to theprevention of water uptake into the rod. The more tightly packed rodsalso slowed the diffusion of CXB from the formulation by creatinglocally saturated regions of CXB within the rod, slowing the dissolutionand release.

Example 9. Measurements of Diameter, Density and In Vitro Experiments on0.5 mm Celecoxib Loaded Silk Fibroin Rods

As seen in the experiments on the 1 mm silk rods, the diameter of the0.5 mm silk-fibroin rods was measured using digital calipers. The rodswere cut to 1 cm lengths to standardize release, and the weights of therods were recorded. The densities of the rods were calculated for eachformulation. The rods were placed into 45 mL of 1× phosphate buffer, pH7.4, 0.3% (v/v) Polysorbate-80 (from Croda, Snaith UK), and 0.05% (w/v)sodium azide (from Fisher Chemical, Waltham Mass.). This buffer ensuredthat the release was conducted under sink conditions (≥5× saturatedsolubility). A suspension of celecoxib (CXB) (from Cipla. Miami Fla.)containing 800 μg CXB was used as a control. The samples were incubatedat 37° C. with gentle shaking. 1 mL of the release medium was taken ateach timepoint (typically 1, 4, and 7 days and then weekly thereafter).The release medium was then analyzed via UPLC at 260 nm to determine CXBconcentration. The data from the experiment was summarized in Table 10.Extraction controls were run to determine CXB loading in the rods.Pre-weighed, 1 cm lengths of the rods were placed into 2 mL of 100%methanol, vortexed, and sonicated. The samples were left to shakeovernight at room temperature. The methanol was then analyzed for CXBloading via HPLC.

TABLE 10 Precise diameter, density, and loading percentages of 0.5 mmsilk-fibroin rods (480 mb; 0.5 mm; 40% st; 100 mgsf; 200 mgcxb; lyo;33.3% sf; 66.7% cxb) Ratio Ratio Theoretical Actual Standard SampleCXB:SF CXB:SF Diameter Density CXB % CXB % Dev. of No. TheoreticalActual (mm) (g/mL) (w/w) (w/w) CXB % 8-65-6 2 1.0 0.43 1.2 66.7 48.9 3.2

The release of CXB was monitored as described over a period of 77 days,as seen in Table 11 and Table 12. The data demonstrated near-zero-orderrelease kinetics. The CXB suspension was completely released after 1day. The rod formulation, however, displayed very extended release. Theinitial burst from the rod was only 12.9% with near zero-order releaseout to 21 days. After 21 days, the release rate slowed even more,allowing for a second zero-order segment of release out to completion atabout 70 days. After day 70, no additional API was released. In Table11, “Std. Dev.” refers to standard deviation.

TABLE 11 In vitro release kinetics of celecoxib from 0.5 mm silk-fibroin rods; average cumulative percentage of API released AverageCumulative % Released CXB Std. Dev. 8-65-6 Day Suspension suspension8-65-6 Std. Dev. 0 0 0 0 0 1 111.8 0.8 12.9 1.3 2 110.9 0.7 18.7 1.3 7113.8 0.8 43.7 2.4 10 — — 57.9 2 7 14 — — 65.4 2.9 21 — — 84.7 4.9 28 —— 92.5 5.3 35 — — 95.1 5.4 42 — — 102.1 5.5 49 — — 113.9 5.9 56 — —118.9 5.6 63 — — 124.0 5.0 70 — — 117.8 4.7 77 — — 117.2 4.4

TABLE 12 Daily percentage of celecoxib released for rods of differentdiameters Measured Initial rod Sample Initial Daily % burst:Dailydiameter No. Sample Name Burst % Released release (mm) 8-65-6 480 mb;0.5 mm; 40% st; 12.9 1.8 7.2 0.43 100 mgsf; 200 mgcxb; lyo; 33.3% sf;66.7% cxb 8-58-8 480 mb; 1 mm; 40% st; 6.2 0.9 6.9 0.91 100 mgsf; 200mgcxb; lyo; 33.3% sf; 66.7% cxb

The data from this experiment suggested that the rate of release oftherapeutic, CXB, was inversely related to the diameter of the silkrods. The daily release percentage of CXB, as well as the ratio of theinitial burst to the daily release percentage and other rod parameters,is shown in Table 12. The daily percentage of CXB released for sample8-65-6, which was calculated for 63 days, was 1.8%. The corresponding 1mm silk rods (Sample 8-58-8), as seen in 1 mm silk rod experiments, were33.3% (w/w) SF, 66.7% (w/w) CXB, and had a 480-minute boil. These 1 mmsilk rods released 0.9% of the loaded CXB per day. The almost two-folddifference between the 1 mm and 0.5 mm silk rods suggested that thetherapeutics were released more quickly from rods with a smallerdiameter. This difference was also observed in the initial burst of drugrelease; however, the ratio between the initial burst and the dailyrelease percentage remained consistent regardless of rod diameter. The 1mm silk rods had an initial burst of 6.2%, while the corresponding 0.5mm rods had an initial burst of 12.9%. The changes in initial burst anddaily release percentage were likely due to the greater surface area tovolume ratio in the rods of smaller diameter. In the narrower rods,water penetration and diffusion lengths were shorter, which lead to thefaster releasing effect. These narrower rods could be injected through a21-22G needle (standard for intravitreal injection devices), making themappropriate for intraocular delivery.

It should be noted that the actual CXB loading of 1 mm rods was higherthan that of the 0.5 mm silk rods. This higher loading could alter therate of CXB release between rods of the same theoretical formulation.Furthermore, the experiments for the 1 mm silk rods were carried outover a period of 126 days, which was longer than the experiments for the0.5 mm rods. The release of CXB may decrease over longer periods oftime, and the potential change in rate over time may alter the averagedaily percentage released.

Example 10. Comparison of Silk Fibroin Rods Prepared Via LyophilizationVs Oven Drying

The silk yarn was purchased from Jiangsu SOHO International Group(Jiangsu, China). Lithium bromide and phosphate buffer saline werepurchased from Sigma Aldrich (St. Louis, Mo.). The potassium phosphatemonobasic and potassium phosphate dibasic were purchased from SigmaAldrich Fine Chemicals (SAFC) (St. Louis, Mo.). The sodium carbonate andthe sodium azide were purchased from Fisher Chemical (Waltham, Mass.).The celecoxib (CXB) was purchased from Cipla (Miami, Fla.).

Silk Fibroin Isolation

The silk yam was degummed at 100° C. for either 120 or 480 minutes in0.02 M sodium carbonate solution to remove sericin and modify themolecular weight. The total boiling time was discussed in terms ofminute boil, or “mb.” Longer boiling times produced silk fibroin withlower average molecular weights. The objective of this experiment was todetermine any difference in the release rate of the API between the silkrods prepared via lyophilization and the silk rods prepared via ovendrying. Silk-fibroin (Jiangsu SOHO) was isolated as described in thepreparation of the silk fibroin rods with no additives. Briefly, Silkyarn, purchased from Jiangsu SOHO International Group, was degummed toremove sericin. 30 grams of cut silk yarn were boiled at 100° C. in 3 Lof deionized (DI) water with 0.02 M sodium carbonate with stirring. Theyam was then transferred to a new boiling 0.02 M sodium carbonateaqueous solution and boiled at 100° C. for additional time withstirring. The fibroin was then placed in DI water at 60-70° C. for 20minutes with stirring, and then rinsed with clean DI water. This processwas repeated 3 times. The fibroin was placed in clean DI water, stirredfor 20 minutes, then rinsed with clean DI water, and this process wasrepeated for a total of 3×20 min.-rinse cycles.

The fibroin was dried overnight, weighed, and dissolved at 20% (w/v) ina 9.3 M aqueous solution of lithium bromide (from Sigma-Aldrich, St.Louis, Mo.) for 5 hours at 60° C. The resulting fibroin solution wasdialyzed against water at 4° C. in a 50 kDa regenerated cellulosedialysis tubing for 48 hours, with 6 water changes to remove the excesssalt. The conductivity was recorded after each water change with adigital quality tester. When the conductivity was under 5 ppm, thefibroin solution was determined to be ready. The solution was thencentrifuged for 20 minutes at 9,000 RPM and 4° C. to remove insolubleparticles. Solutions were diluted to a final concentration of 3% (w/v)in 10 mM phosphate buffer, pH 7.4, filtered through a 0.22 μm filter,frozen in liquid nitrogen, and lyophilized for 72 hours.

Silk Fibroin Rod Preparation

Lyophilized silk fibroin was reconstituted to either 20, 30, or 40%(w/v) with DI water. The desired amount of CXB was weighed into 4 mLglass vials. 250 μL of stock fibroin solution was then added to eachvial accordingly. The fibroin and CXB was mixed both manually using aspatula and with a vortex. This mixture was then transferred to a 1 mLsyringe using the spatula and extruded into 2×10 cm lengths of 500 μm IDpolytetrafluoroethylene (PTFE) tubing (from Van Waters and Rogers (VWR),PA, USA). The tubing was then sealed on both ends using Parafilm andincubated at 37° C. to induce gelation. The lengths of tubing were cutinto 2 cm sections. Half of the sections were dried for 48 hours in anoven at 60° C. The other half were frozen at −80° C. and lyophilized.Rods were stored at 4° C. prior to use.

The samples, shown in Table 13, are named by the process used to prepareand formulate each silk rod. For example, the sample named “480 mb; 0.5mm; 40% st; 100 mgsf; 100 mgcxb; lyo; 50% sf; 50% cxb” refers to a silkfibroin rod prepared from silk degummed with a 480-minute boil, anextrusion with a 0.5 mm diameter, a preparation from a 40% stocksolution of silk fibroin, a preparation from 100 mg of silk fibroin, apreparation from 100 mg of celecoxib, lyophilization, a theoretical w/wpercentage of 50% silk fibroin, and a theoretical w/w percentage of 50%celecoxib. Samples prepared via oven drying were labeled with “oven”.Some samples were prepared with silk fibroin degummed with a 120-minuteboil (120 mb). The final rods contained trace amounts of potassiumphosphate buffer (with potassium phosphate dibasic and potassiumphosphate buffer monobasic). In Table 13, “Std. Dev.” refers to standarddeviation.

TABLE 13 Theoretical and experimental loading percentages for oven-driedand lyophilized 0.5 mm silk-fibroin rods Actual Std. Silk Stock Conc.Silk- Actual Dev. of Prep Phosphate of Silk for Fibroin CXB CXB BoilBuffer Sample Sample Formulation Drying Final % Final % Final % TimeConc. No. Name (w/v %) Method (w/w) (w/w) (w/w) (min) (mM) 177-1A 480mb; 40 Lyophilized 62.30 37.70 0.52 480 95.2 0.5 mm; 40% st; 100 mgsf;100 mgcxb; lyo; 50% sf; 50% cxb 177-1B 480 mb; 40 Oven 61.54 38.46 0.08480 95.2 0.5 mm; 40% st; 100 mgsf; 100 mgcxb; oven; 50% sf; 50% cxb177-2A 480 mb; 40 Lyophilized 53.14 46.86 0.70 480 83.3 0.5 mm; 40% st;100 mgsf; 150 mgcxb; lyo; 40% sf; 60% cxb 177-2B 480 mb; 40 Oven 53.2746.73 1.19 480 83.3 0.5 mm; 40% st; 100 mgsf; 150 mgcxb; oven; 40% sf;60% cxb 177-4A 480 mb; 40 Lyophilized 45.61 54.39 0.92 480 66.7 0.5 mm;40% st; 100 mgsf; 250 mgcxb; lyo; 28.6% sf; 71.4% cxb 177-4B 480 mb; 40Oven 45.72 54.28 0.93 480 66.7 0.5 mm; 40% st; 100 mgsf; 250 mgcxb;oven; 28.6% sf; 71.4% cxb 177-6A 480 mb; 30 Lyophilized 46.03 53.97 1.82480 55.6 0.5 mm; 30% st; 75 mgsf; 200 mgcxb; lyo; 27.3% sf; 72.7% cxb177-6B 480 mb; 30 Oven 45.27 54.73 1.01 480 55.6 0.5 mm; 30% st; 75mgsf; 200 mgcxb; oven; 27.3% sf; 72.7% cxb 177-7A 120 mb; 20 Lyophilized43.35 56.65 2.97 120 37 0.5 mm; 20% st; 50 mgsf; 200 mgcxb; lyo; 20% sf;80% cxb 177-7B 120 mb; 20 Oven 39.71 60.29 0.26 120 37 0.5 mm; 20% st;50 mgsf; 200 mgcxb; oven; 20% sf; 80% cxb 177-8A 120 mb; 30 Lyophilized42.23 57.77 4.08 120 55.6 0.5 mm; 30% st; 75 mgsf; 200 mgcxb; lyo; 27.3%sf; 72.7% cxb 177-8A 120 mb; 30 Oven 42.25 57.75 3.87 120 55.6 0.5 mm;30% st; 75 mgsf; 200 mgcxb; oven; 27.3% sf; 72.7% cxb 177-9A 120 mb; 40Lyophilized 48.46 51.54 0.48 120 74.1 0.5 mm; 40% st; 100 mgsf; 200mgcxb; lyo; 33.3% sf; 66.7% cxb 177-9B 120 mb; 40 Oven 48.93 51.07 3.46120 74.1 0.5 mm; 40% st; 100 mgsf; 200 mgcxb; oven; 33.3% sf; 66.7% cxb

In Vitro Release

The rods were cut to 1 cm lengths to standardize release, and theweights of the rods were recorded. In triplicate, a 1 cm segment of rodwas weighed into a 50 mL conical tube. 45 mL of release medium(phosphate buffered saline, 0.3% Polysorbate-80, and 0.05% sodium azide)was added to each tube. We had previously shown that this media wouldensure sink conditions (≥5×CXB solubility) are maintained throughout thestudy. The tubes were incubated at 37° C. with shaking. 1 mL of therelease media was collected from each sample at days 1,4, 7, 10, 14, andweekly thereafter and replaced with fresh media. Release media wasanalyzed for CXB concentration by HPLC-UV at 260 nm.

Controls were prepared by weighing 1 cm of each formulation intriplicate in separate glass vials. Methanol was added to each vial.Samples were vortexed, sonicated, and placed on a shaker at roomtemperature for 24 hours. The supernatant was analyzed by HPLC todetermine CXB loading (mg/g), as seen in Table 13. CXB loadedsilk-fibroin rods were prepared with loadings ranging from 38-60% (w/w).Drying method did not have an impact on the drug loading, suggestingthat the drug was stable through the 60° C. treatment. The releasekinetics of both the lyophilized and the oven dried silk rods were shownin Table 14A, Table 14B, and Table 15. All samples showed zero percent(% i)API release on day zero. The rods demonstrated near zero-orderkinetics of API release.

TABLE 14A In vitro release kinetics of celecoxib from 0.5 mmsilk-fibroin rods, lyophilized vs. oven dried; average cumulativepercentage of API released Lot Day No. 1 3 7 10 14 21 28 35 42 49 56 63177-1A 13.6 27.8 51.5 58.9 70.5 80.2 92.2 105.9 112.3 110.7 114.8 —177-1B 10.1 21.6 41.0 47.4 57.2 66.3 77.3 90.3  97.6 99.8 108.9 112.2177-2A 15.4 32.2 59.6 66.9 78.7 87.9 97.3 105.9 108.0 104.8 — — 177-2B12.1 25.4 47.4 54.7 64.8 73.6 83.5 95.0 100.6 99.9 106.0 — 177-4A 13.025.8 50.0 55.7 66.4 75.3 87.5 97.8 106.9 106.7 110.7 — 177-4B 14.2 30.557.6 65.2 76.9 85.9 96.1 108.1 112.4 108.2 — — 177-6A 18.4 35.7 63.370.9 82.6 90.4 96.0 102.9 103.5 99.5 — — 177-6B 16.2 33.8 66.2 72.8 85.794.4 100.0 107.4 108.4 104.1 — — 177-7A 23.7 46.6 83.6 93.7 108.5 112.3111.0 — — — — — 177-7B 15.7 34.3 67.3 76.6 90.9 100.4 104.9 108.8 108.6104.3 — — 177-8A 14.5 31.6 57.7 66.3 78.5 87.8 97.2 106.3 106.5 102.4 —— 177-8B 15.8 31.9 58.5 66.2 77.9 87.1 96.8 106.2 106.8 102.4 — — 177-9A14.1 28.8 51.6 58.1 68.6 76.5 86.0 97.3 102.2 99.5 — — 177-9B 13.4 27.448.9 54.8 64.7 72.2 81.4 92.5  97.9 97.6 101.9 — 177-10 106.2 106.2110.9 — — — — — — — — —

TABLE 14B Standard deviation of the In vitro release kinetics ofcelecoxib from 0.5 mm silk- fibroin rods, lyophilized vs. oven dried;cumulative percentage of API released Lot Day No. 0 1 3 7 10 14 21 28 3542 49 56 63 177-1A 0 1.1 2.2 4.0 4.9 5.5 6.9 8.2 10.2  11.2  10.3  8.0 —177-1B 0 0.5 1.2 1.7 2.0 2.1 2.3 2.6 4.2 4.3 4.1 4.2 4.1 177-2A 0 3.05.6 9.6 10.2 11.3 11.8 12.1 10.3  7.6 6.3 — — 177-2B 0 2.6 3.9 6.2 6.97.4 7.8 8.0 7.8 5.7 2.7 1.0 — 177-4A 0 0.3 0.8 0.9 1.0 1.2 1.7 1.6 2.61.9 1.6 1.5 — 177-4B 0 0.7 1.2 2.6 3.2 3.9 3.5 3.7 4.3 4.4 3.9 — —177-6A 0 4.1 7.2 10.3 10.2 10.7 9.0 6.6 8.1 8.9 8.4 — — 177-6B 0 2.8 6.712.4 13.1 14.8 13.3 7.7 4.2 3.2 3.3 — — 177-7A 0 4.7 5.4 7.7 7.2 6.7 3.94.0 — — — — — 177-7B 0 0.3 1.2 3.1 3.4 4.1 3.6 2.7 2.4 2.4 2.4 — —177-8A 0 0.1 2.2 3.4 3.8 3.9 3.5 3.1 2.9 2.9 2.7 — — 177-8B 0 1.6 3.13.3 2.9 3.2 3.0 3.7 5.1 5.5 5.1 — — 177-9A 0 1.7 3.1 4.5 4.5 5.0 5.0 4.23.4 1.9 1.0 — — 177-9B 0 0.7 1.0 1.9 1.9 1.5 1.5 0.9 0.6 0.3 1.0 2.3 —177-10 0.0 0.4 0.3 0.4 — — — — — — — — —

All CXB loaded rod formulations exhibited biphasic release. Initialzero-order release PG-9T from 1-10 days and a second zero-order profilefrom 10 days to completion. The rods reached complete release between 14and 56 days.

In many of the samples subjected to the 480 mb degumming process, theinitial burst of API release, determined as the total w/w percentage ofCXB released in one day, was smaller for the oven dried silk rods thanthe lyophilized silk rods. For many rods prepared under identicalconditions except for drying, the oven dried rods released between 5 and35% less API during the initial burst than their lyophilizedcounterparts. This difference, shown in Table 15, was determined as thepercent error between the initial bursts of the oven dried andlyophilized rods prepared under otherwise identical conditions.

TABLE 15 Analysis of initial burst percentages of oven dried and freezedried 0.5 mm rods Initial burst (% API Sample released by Difference No.Sample Name mass) by % 177-1A 480 mb; 0.5 mm; 40% st; 100 mgsf; 100mgcxb; lyo; 50% sf; 50% cxb 13.6 25.8 177-1B 480 mb; 0.5 mm; 40% st; 100mgsf; 100 mgcxb; oven; 50% sf; 50% cxb 10.1 — 177-2A 480 mb; 0.5 mm; 40%st; 100 mgsf; 150 mgcxb; lyo; 40% sf; 60% cxb 15.4 21.4 177-2B 480 mb;0.5 mm; 40% st; 100 mgsf; 150 mgcxb; oven; 40% sf; 60% cxb 12.1 — 177-4A480 mb; 0.5 mm; 40% st; 100 mgsf; 250 mgcxb; lyo; 28.6% sf; 71.4% cxb13.0 −9.6 177-4B 480 mb; 0.5 mm; 40% st; 100 mgsf; 250 mgcxb; oven;28.6% sf; 14.2 — 71.4% cxb 177-6A 480 mb; 0.5 mm; 30% st; 75 mgsf; 200mgcxb; lyo; 27.3% sf; 72.7% cxb 18.4 12.0 177-6B 480 mb; 0.5 mm; 30% st;75 mgsf; 200 mgcxb; oven; 27.3% sf; 72.7% cxb 16.2 — 177-7A 120 mb; 0.5mm; 20% st; 50 mgsf; 200 mgcxb; lyo; 20% sf; 80% cxb 23.7 33.8 177-7B120 mb; 0.5 mm; 20% st; 50 mgsf; 200 mgcxb; oven; 20% sf; 80% cxb 15.7 —177-8A 120 mb; 0.5 mm; 30% st; 75 mgsf; 200 mgcxb; lyo; 27.3% sf; 72.7%cxb 14.5 −8.9 177-8B 120 mb; 0.5 mm; 30% st; 75 mgsf; 200 mgcxb; oven;27.3% sf; 72.7% cxb 15.8 — 177-9A 120 mb; 0.5 mm; 40% st; 100 mgsf; 200mgcxb; lyo; 33.3% sf; 66.7% cxb 14.1  5.2 177-9B 120 mb; 0.5 mm; 40% st;100 mgsf; 200 mgcxb; oven; 33.3% sf; 13.4 — 177-10 CXB suspension 106.2N/A

Samples 177-6 (A and B, both oven dried and lyophilized), were preparedin a manner identical to that of samples 177-8 (A and B, both oven driedand lyophilized), except for the boiling time of the silk fibroin. Aspreviously stated, an increase in the boiling time reduces the molecularweight of the silk fibroin. Consequently, these samples allowed for thedirect comparison of rods prepared identically with different molecularweights of silk fibroin. The lyophilized samples with a higher molecularweight (120 mb) exhibited an initial burst that was 21.1% less than thelyophilized samples prepared with a lower molecular weight (480 mb).Meanwhile, the oven dried samples with a higher molecular weight (120mb) exhibited an initial burst that was 2.5% less than the oven driedsamples prepared with a lower molecular weight (480 mb).

The daily release percentages were also compared to the initial burstpercentages. The daily release percentages, as well as the ratio of theinitial burst percentages to the daily release percentages, werecalculated from the data from the in vitro release experiments, andthese data were displayed in Table 16. The daily release percentageswere calculated for the first 49 days of the study.

TABLE 16 Daily percentage of celecoxib release for rods of differentdrying methods and different boiling times Initial Ratio Ratio InitialDaily burst:Daily CXB:SF CXB:SF Lot Sample Name Burst % Release %release Theoretical Actual 177-1A 480 mb; 0.5 mm; 40% st; 13.6 2.1 6.31.0 0.6 100 mgsf; 100 mgcxb; lyo; 50% sf; 50% cxb 177-1B 480 mb; 0.5 mm;40% st; 10.1 1.9 5.2 1.0 0.6 100 mgsf; 100 mgcxb; oven; 50% sf; 50% cxb177-2A 480 mb; 0.5 mm; 40% st; 15.4 2.0 7.9 1.5 0.9 100 mgsf; 150 mgcxb;lyo; 40% sf; 60% cxb 177-2B 480 mb; 0.5 mm; 40% st; 12.1 1.9 6.4 1.5 0.9100 mgsf; 150 mgcxb; oven; 40% sf; 60% cxb 177-4A 480 mb; 0.5 mm; 40%st; 13.0 2.0 6.4 2.5 1.2 100 mgsf; 250 mgcxb; lyo; 28.6% sf; 71.4% cxb177-4B 480 mb; 0.5 mm; 40% st; 14.2 2.1 6.9 2.5 1.2 100 mgsf; 250 mgcxb;oven; 28.6% sf; 71.4% cxb 177-6A 480 mb; 0.5 mm; 30% st; 18.4 1.8 10.32.7 1.2 75 mgsf; 200 mgcxb; lyo; 27.3% sf; 72.7% cxb 177-6B 480 mb; 0.5mm; 30% st; 16.2 1.9 8.5 2.7 1.2 75 mgsf; 200 mgcxb; oven; 27.3% sf;72.7% cxb 177-7A 120 mb; 0.5 mm; 20% st; 23.7 3.7 6.4 4.0 1.3 50 mgsf;200 mgcxb; lyo; 20% sf; 80% cxb 177-7B 120 mb; 0.5 mm; 20% st; 15.7 1.98.2 4.0 1.5 50 mgsf; 200 mgcxb; oven; 20% sf; 80% cxb 177-8A 120 mb; 0.5mm; 30% st; 14.5 1.9 7.5 2.7 1.4 75 mgsf; 200 mgcxb; lyo; 27.3% sf;72.7% cxb 177-8B 120 mb; 0.5 mm; 30% st; 15.8 1.9 8.2 2.7 1.4 75 mgsf;200 mgcxb; oven; 27.3% sf; 72.7% cxb 177-9A 120 mb; 0.5 mm; 40% st; 14.11.9 7.5 2.0 1.1 100 mgsf; 200 mgcxb; lyo; 33.3% sf; 66.7% cxb 177-9B 120mb; 0.5 mm; 40% st; 13.4 1.8 7.4 2.0 1.0 100 mgsf; 200 mgcxb; oven;33.3% sf; 66.7% cxb 177-10 CXB Suspension 106.2 N/A N/A N/A N/A

Oven dried rods showed slower release than the lyophilized rods, withlower initial burst percentages, however they also showed similarbiphasic release profiles. The second phase of release, however, wasdelayed from 10 to 14 days when the rods were oven dried. The completerelease of CXB ranged from 35 to greater than 63 days and followed thesame trends as the lyophilized rods (rates increasing with increasingCXB:silk ratio). This slower release of the oven-dried rods was mostlikely due to increased beta-sheet content of the silk-fibroin as wellas decreased porosity of the rods. Both factors would make the rods morehydrophobic, slowing water uptake and decreasing diffusion of CXB.

The data also revealed that the (w/w) ratio of API to silk fibroin wasdirectly proportional to the initial burst percentage. In the context ofthe 0.5 mm silk fibroin rods, lower initial burst percentagescorresponded with lower ratios of CXB to silk fibroin, while higherinitial burst percentages corresponded to higher ratios of CXB to silkfibroin. The daily release percentage of the 0.5 mm rods also increasedas the ratio of CXB to silk fibroin increased. As the drug loadingincreased and silk-fibroin concentration decreased, the release ratesincreased. This suggested that the silk-fibroin was controlling releaseand that release rates could be tuned using this variable.

The measured and calculated parameters of the rods were also examined inthe context of silk fibroin boiling time and molecular weight, bycomparing the experimental results from rods of lot numbers 177-6 (A andB) and 177-8 (A and B). As stated previously, the rods from thesepreparations were identical except for the boiling time, and thereforethe molecular weight, of the silk fibroin. The ratio of the initialburst percentages to the daily release percentages was lower for rodsprepared from higher molecular weight silk fibroin; this result waslikely due to the observed lower initial burst percentage with silk rodsof higher molecular weight silk fibroin. Meanwhile, the daily releasepercentages differed by only 0.1% between the freeze-dried rods withlower and higher molecular weights; the daily release percentages ofthese samples were 1.8% and 1.9% respectively. The daily releasepercentages did not change between oven dried samples of lower andhigher molecular weight; the daily release percentage for those sampleswas 1.9%. As a result, it was concluded that the boiling time, andconsequently the molecular weight, of the silk fibroin did not affectthe daily release percentages of the silk fibroin rods. These in vitrocharacterizations displayed that release from these formulations wasindependent of the silk-fibroin molecular weights assessed.

Example 11. In Vivo Study of Silk Fibroin Rods with Celecoxib in anAnimal Model

As with the hydrogels without celecoxib (CXB), all buffers and stocksolutions were prepared under sterile conditions unless otherwiseindicated. All formulations were prepared with SOHO silk yarn. Thepoloxamer-188 was from Sigma-Aldrich (St. Louis, Mo.), while the PEG4kDa was from Clariant, Charlotte N.C. Multiple preparations of the sameformulations may be used in the study and overall analysis.

Preparation of Celecoxib Experimental Controls

As seen in the hydrogels formulated with CXB, a 27.8% suspension ofcelecoxib (CXB) was prepared from 4.15 g dry heat treated (DHT) CXB(from Cipla, Miami Fla.) in 10.78 mL of 0.79% Polysorbate-80 (fromCroda. Snaith UK) and mixed until homogenous. To prepare the 10% CXBsuspension as a control, a 1.789 mL fraction of the 27.8% CXB suspensionwas diluted to 5 mL via the addition of 0.349 mL 315 mM PB (pH=7.4),0.158 mL of 200 mg/mL NaCl, and DI water. The resulting 10% CXBsuspension was immediately aliquoted into 0.2 mL fractions in I ccsyringes so that it remained homogenous, and the fractions were storedon ice until subsequent injection. To prepare the 0.2% CXB suspension asan additional control, a 0.18 mL fraction of the 10% CXB solution wasdiluted with 0.686 mL of 315 mM PB (pH=7.4), 0.31 mL of 200 mg/mL NaCl,2.468 mL of 0.79% Polysorbate −80, and DI water to a final volume of 10mL. The suspension was mixed until homogenous, aliquoted into 0.2 mLfractions, and stored on ice until use.

Preparation of Silk Fibroin Materials for Injection

The efficacy of the silk rods was compared to that of silk fibroinhydrogels. Both unadulterated silk fibroin hydrogels and silk fibroinhydrogels with 10% CXB were prepared as experimental controls. Allprocesses were performed under aseptic conditions using pre-sterilizedmaterials. To prepare the unadulterated silk fibroin hydrogel (sample3B) 300 mg of 480 mb silk fibroin were brought up in 3.342 mL 0.6%Polysorbate −80, 0.383 mL of 315 mM phosphate buffer (pH=7.4), and 0.246mL DI water. To prepare the 10% CXB hydrogel (sample 4B), 300 mg of 480mb silk fibroin were brought up in 3.589 mL of the 27.8% CXB suspensionand 0.381 mL of 315 mM PB (pH=7.4). Both the solutions for the hydrogelsamples were incubated at room temperature and mixed for 30 minutesuntil homogenous. Each mixture was then aliquoted into 3.41 mL fractionsin 10 cc syringes. The samples in Table 17 are named by the process usedto prepare and formulate each hydrogel. For example, in the sample named480 mb; hyd; 27.8% cxbst; 3% SFf; 10% CXBf; 10% P188f, “480 mb” refersto silk degummed with a 480-minute boil, “hyd” refers to the formulationof the sample as a hydrogel, “27.8% cxbst” refers to a preparation froma stock solution of 27.8% of celecoxib, “3% SFf” refers to a formulationwith 3% (w/v) silk fibroin, “10% CXBf” refers to a formulation with 10%(w/v) celecoxib, and “10% PI88f” refers to a formulation with 10% (w/v)poloxamer 188. The sample named “480 mb; 0.5 mm, 40% st; 100 mgsf; 200mgcxb; lyo; 33.3% sf; 66.7% cxb” refers to a silk fibroin rod preparedfrom silk degummed with a 480-minute boil, an extrusion with a 0.5 mmdiameter, a preparation from a 40% stock solution of silk fibroin, apreparation from 100 mg of silk fibroin, a preparation from 200 mg ofcelecoxib, lyophilization, a theoretical w/w percentage of 33.3% silkfibroin, and a theoretical w/w percentage of 66.7% celecoxib. Allsuspension and gel formulations contained 0.2% polysorbate-80 and 22 mMphosphate buffer. The 1.4% CXB suspension contained 6.34 mg/mL NaCl. The10% CXB suspension contained 6.32 mg/mL NaCl. Both hydrogels contained5.94 mg/mL NaCl. The rods contained 74.1 mM phosphate buffer.

TABLE 17 Descriptions of samples for in vivo experiments of silk fibroinrods with celecoxib Silk- Silk- Fibroin Fibroin Excipient Boil Conc.Conc. Sample Name Description Time (%) Excipient (%) 1.4% CXB control1.4% CXB Suspension — — — — 10% CXB control 10% CXB Suspension — — — —480 mb; hyd; 0% cxbst; 3% 480 mb; 10% P188 480 3 P188 10 3% SFf; 0%CXBf; 10% P188f 480 mb; hyd; 3% 480 mb; 10% 480 3 P188 10 27.8% cxbst;3% SFf; P188; 10% CXB 10% CXBf; 10% P188f 480 mb; 0.5 mm; 20% 480 mb;40% 480 20 — — 40% st; 100 mgsf; CXB Rods 200 mgcxb; lyo; 33.3% sf;66.7% cxb

An excipient solution was prepared from 13.05 mL of stock 20% P188,0.777 mL of 200 mg/mL NaCl, and 1.173 mL of DI water. This excipientsolution was prepared in 10 cc syringes in 4.59 mL aliquots. For eachsample, the syringe of the representative silk fibroin solution wasconnected to a syringe of its designated excipient solution via a BBraun fluid dispensing connector. The contents of the syringes were thenmixed until homogenous. The resulting samples were incubated on arotator for 24 hours at 37° C. and then separated into 0.2 mL aliquotsin 1 cc syringes. The pH values of the samples were measured with aglass pH probe. Samples were stored at 4° C., as needed. Formulations ofthe hydrogels contained 1.04% (w/v) sodium chloride, 0.2% (w/v)Polysorbate-80, and 22 mM phosphate buffer at pH=7.4 for theP188-containing hydrogels. Some formulations comprised 10% P188, 10%CXB, and 10.4 mg/mL sodium chloride at a pH of 7.4.

The silk fibroin rods were prepared as described in the preparation of0.5 mm silk fibroin rods. Briefly, 600 mg of 480 mb silk fibroin weredissolved in 0.900 mL of DI water. 0.591 mL of the resulting solutionwas then used to bring up 473 mg of CXB, vortexed, and mixed. Themixture of silk fibroin and CXB was further mixed back and forth througha syringe connector until the mixture was homogenous. The mixture wasthen capped with a 27-gauge, 0.5 inch, needle and extruded into 10 cmlengths of 0.02″ ID PEEK tubing. The tubing was cut into 2 cm pieces andincubated overnight at 37° C. under sterile conditions. The rods werethen removed from the tubing, frozen, and lyophilized overnight.Lyophilized rods were stored at 4° C. until injection. Therod-containing sample is named by the process used to prepare andformulate each silk rod. For example, the sample named “480 mb; 0.5 mm;40% st; 100 mgsf; 200 mgcxb; lyo; 33.3% sf; 66.7% cxb” refers to a silkfibroin rod prepared from silk degummed with a 480-minute boil, anextrusion with a 0.5 mm diameter, a preparation from a 40% stocksolution of silk fibroin, a preparation from 100 mg of silk fibroin, apreparation from 200 mg of celecoxib, lyophilization, a theoretical w/wpercentage of 33.3% silk fibroin, and a theoretical w/w percentage of66.7% silk fibroin. CXB loaded rods were cut to 1 cm lengths andpreloaded into 21G, 1″ needles. The final formulations of the rods alsocontained trace amounts of potassium phosphate buffer (phosphate buffermonobasic and phosphate buffer dibasic). In vitro release profile ofhydrogel for in vivo experiments

The silk fibroin hydrogels were subject to the in vitro releaseexperiments used to analyze silk hydrogels of varying concentration andsilk fibroin boiling time. Briefly, In triplicate, 50 mg of eachformulation were weighed into half of a #4 gelatin capsule (MyHerbar,Dallas Tex.). Capsules were added to 45 mL of release medium (Ixphosphate buffered saline, 2% polysorbate-80, and 0.05% sodium azide).It had previously been shown that the solubility of celecoxib in thisrelease media is 850 μg/mL. 45 mL of this release media allowed for 38mg CXB solubility. This media will ensure sink conditions (greater thanor equal to 5 times the CXB solubility) are maintained throughout thestudy. The tubes were incubated at 37° C. with shaking. 1 mL of therelease media was collected from each sample at days 1, 4, 7, 10, 14 andweekly thereafter and replaced with fresh media. At each timepoint, thetubes were placed upright for at least 15 minutes to allow theformulation to settle prior to taking the sample. Release media wasanalyzed by HPLC (Agilent 1290 HPLC system) at 260 nm

Controls were prepared at Day 0 by weighing 50 mg of each formulation intriplicate in separate glass vials. Methanol was added to each sample toextract CXB. Samples were placed on a shaker at room temperature for 24hrs. The supernatant was analyzed by HPLC to determine CXB loading.

The plot of the cumulative percentage of API released over time can beseen in FIG. 4. The release of the API from the hydrogel was much slowerthan the CXB suspension, which served as a control. The release of APIfrom the hydrogel followed first order kinetics, and it occurred overthe span of 1 month. The initial burst was approximately 40%.

In Vitro Release Profile of Rods for In Vivo Experiments

The silk fibroin rods were subject to the in vitro release experimentsused to analyze silk fibroin rods of both 1 mm and 0.5 mm diameterloaded with CXB. Briefly, 1 cm segments of rod were weighed into 50 mLconical tubes. 45 mL of release medium (phosphate buffered saline, 0.3%polysorbate-80, and 0.05% sodium azide) was added to each tube. It hadpreviously been shown that this media would ensure sink conditions(greater than or equal to 5 times CXB solubility) were maintainedthroughout the study The tubes were incubated at 37° C. with shaking. ImL of the release media was collected from each sample at days 1, 4, 7,10, 14 and weekly thereafter and replaced with fresh media.

Controls were prepared by weighing 1 cm of each formulation intriplicate in separate glass vials. Methanol was added to each vial.Samples were vortexed, sonicated, and placed on a shaker at RT for 24hrs. The supernatant was analyzed by HPLC to determine CXB loading(mg/g).

Release media was analyzed for CXB concentration by HPLC-UV (Agilent1290 HPLC system) 260 nm. The average cumulative percentage of APIreleased over time was listed in Table 18 and FIG. 4. The release of CXBfollowed near zero-order kinetics. Cumulative percent released wascalculated with a daily standard curve unless otherwise indicated. InTable 18, “Std. Dev.” refers to standard deviation.

TABLE 18 In vitro release kinetics of celecoxib from 480 mb; 0.5 mm; 40%st; 100 mgsf; 200 mgcxb; lyo; 33.3% sf; 66.7% cxb Average Cumulative %Average Released Std. Dev. Cumulative % Std. (Calculated with(Calculated with Day Released Dev. single standard) single standard) 00.0 0 0.0 0 1 12.5 1.2 11.1 1.0 3 26.4 2.0 23.4 1.4 7 50.4 3.0 41.0 2.410 58.0 4.2 50.1 3.5 14 69.5 5.0 60.8 4.4 21 79.0 5.7 74.0 5.1 28 90.45.5 85.4 5.4 35 105.2 5.7 94.4 5.2 42 112.1 5.3 101.2 4.3 49 119.7 4.7103.5 4.0 56 — — 102.9 3.4 63 — — 102.3 3.2

Additional parameters of the silk fibroin rods were also explored inTable 19. First the actual loading of CXB was determined by UPLC to be48.4%, which was slightly higher than the theoretical loadingpercentage. The initial burst percentage, 11.1%, was then analyzed incomparison with the daily release percentage, 1.6%. The ratio of theinitial burst percentage to the daily release percentage was determinedto be 7.1. Overall, the rods were demonstrated to be capable ofreleasing the API, CXB, over a period of 49 days, and this gradualrelease rendered these rods acceptable candidates for in vivo studies.In Table 19, “Std. Dev.” refers to standard deviation.

TABLE 19 Examining the loading and kinetics of 480 mb; 0.5 mm; 40% st;100 mgsf; 200 mgcxb; lyo; 33.3% sf; 66.7% cxb Sample No. 179 Sample Name480 mb; 0.5 mm; 40% st; 100 mgsf; 200 mgcxb; lyo; 33.3% sf; 66.7% cxbTheoretical % CXB (w/w) 66.7 Actual % CXB (w/w) 48.4 Std. Dev. of actualCXB % (w/w) 0.6 Initial Burst % 12.5 Initial Burst % (single standard)11.1 Daily release % 2.2 Daily release % (single standard) 1.6 InitialBurst:Daily release 5.6 Initial Burst:Daily release (single 7.1standard)Administration of 0.5 mm Rods with Celecoxib

New Zealand adult white rabbits were prepared and draped in the usualsterile fashion. Intravitreal injections were made into the left eye(OS) of all rabbits. Right eyes remained as naïve controls. Animals weregiven a pre-anesthetic (Xylazine 1.1 mg/kg IM, Buprenorphine HCl 2-6mcg/kg IM). Animals were then anesthetized with ketamine 22 mg/kg IM.The animals were placed on a heating pad and their vitals weremonitored. The animals were put on inhalation anesthesia (Isoflurane at1.5-2%) with O₂ supplement.

To administer the hydrogels into the intravitreal space, a lid speculumwas inserted into the rabbit's left eye. The conjunctiva was rinsed withBSS solution. Then, the conjunctival sac was prepped with a 5%ophthalmic betadine solution. The hydrogel was then injected into theintravitreal space using a double-plane injection technique. The sclerawas penetrated at 15°-30° then the needle was repositioned to a 45°-60°angle while the sclera was still engaged; the formulation was deliveredand the needle was removed at a 90° angle. Following injection, thecentral retinal artery was examined via indirect ophthalmoscopy toconfirm perfusion and 1-2 drops of betadine solution were added to theconjunctiva prior to removal of the speculum.

To administer the rods, formulations were pre-loaded into sterile 21 g,1″ needle cannulas. Intracannular plungers were fashioned with 28G wirewhich were pre-cut, sterilized, and placed into the needle from the hub.The same sterile and double-plane injection technique was used as forthe hydrogels. The sclera was penetrated at 15°-30°, then the needle wasrepositioned to a 45°-600 angle while the sclera was still engaged; theformulation was delivered. The plunger was depressed, resulting incomplete delivery of the rod into the eye into the intravitreal space.The wire could be pushed until it extended beyond the needle or cannulato ensure complete delivery. The needle was removed at a 90° angle.Following injection, the central retinal artery was examined viaindirect ophthalmoscopy to confirm perfusion and 1-2 drops of betadinesolution were added to the conjunctiva prior to removal of the speculum.When fully injected, the rod was clear from the wall of the eye.

Intraocular Pressure and Biocompatibility after Rod Administration

Intraocular pressure was measured with a Tono-Pen. 7 days after rodadministration, there were no obvious signs of inflammation. Noelevation in intraocular pressure was detected as compared to the naïvecontralateral eyes. There were slightly lower intraocular pressuresdetected in the eyes treated with celecoxib, as seen in Table 20. As aresult, the celecoxib loaded rods reduced the intraocular pressure ofthe treated eye. The analysis of the intraocular pressure was continuedover the course of the study, as seen in Table 20, and multiplepreparations of the same rod formulations were used. The intraocularpressure in the eyes containing the silk fibroin rod did not increaseover the time evaluated.

No adverse clinical findings were noted throughout the course of thestudy. Mild-vitreous hemorrhage was sometimes observed following rodinjection. Similar findings were seen previously with silk-fibroinsolutions and hydrogels with CXB. Additionally, the histopathologyreport indicated that the rods did not induce any inflammation in thevitreous. There was slight infiltration of macrophages into thesilk-fibroin rods, but there were no signs of inflammation or damage inthe remainder of the eye. In addition, normal or lower intraocularpressure was measured 4 months after rod administration. No localinflammation, hemorrhage, or other complications were detected 4 monthsafter administration. Based on these results, intravitreal injections ofsilk-fibroin rods were determined to be well tolerated in rabbits.

TABLE 20 Intraocular pressure measurements at exams with silk fibroinrods (480 mb; 0.5 mm; 40% st; 100 mgsf; 200 mgcxb; lyo; 33.3% sf; 66.7%cxb) Left Eye (Injected) Right Eye (Naïve) Average Average Fold changein IOP Standard IOP Standard IOP Group Day (mmHg) Deviation (mmHg)Deviation (Injected/Naïve) 1 47 9.00 2.00 12.00 0.00 0.75 30 12.33 4.0411.67 5.86 1.06 88 10.75 3.10 11.75 1.50 0.91 111 12.33 4.04 11.00 5.201.12 126 8.00 4.08 10.00 4.90 0.80 169 10.00 1.41 10.50 2.12 0.95Control 30 5.33 3.51 11.33 1.15 0.47 (1.4% CXB solution

Example 12. In Vivo Study of Silk Fibroin Hydrogels in an Animal Model

All buffers and stock solutions were prepared under sterile conditionsunless otherwise indicated. All formulations were prepared with silk yampurchased from SOHO. The silk rods were prepared with a dose of 750 μgof celecoxib (CXB) (from Cipla, Miami Fla.). The poloxamer-188, sodiumchloride, and hydrochloric acid were from Sigma-Aldrich (St. Louis,Mo.), while the PEG4 kDa was from Clariant, Charlotte N.C.Polysorbate-80 was purchased from Croda (Snaith UK). Potassium phosphatemonobasic and potassium phosphate dibasic were purchased from SigmaAldrich Fine Chemical (SAFC, St. Louis Mo.). Phosphate buffered salinewas purchased from Gibco (USA). Multiple preparations of the sameformulations were used.

Preparation of Silk Fibroin Experimental Controls

A phosphate buffer (PB) control was prepared for the in vivoexperiments. PB was aliquoted into 0.4 mL fractions and stored in I ccsyringes. The PBS controls were stored at 4° C. until time of injection.

A CXB suspension was also prepared as an experimental control. CXB wassuspended in an aqueous solution of sodium chloride (Sigma-Aldrich, St.Louis, Mo.), Polysorbate-80 (Croda, Snaith UK), and phosphate buffer.The CXB was homogeneously dispersed using ultrasonication and stored at4° C. prior to injection. The suspension drawn up into 1 cc. syringesjust prior to injection to avoid settling.

Silk fibroin solutions were prepared by boiling raw silk (from JiangsuSOHO) for 120 minutes (herein referred to as “120 mb”) or by boiling for480 minutes (herein referred to as “480 mb”). 120-minute boil results insilk fibroin with a higher molecular weight than the 480-minute boil.Lyophilized silk-fibroin was reconstituted with an aqueous solution ofsodium chloride, Tween-80, and phosphate buffer. The fibroin was allowedto fully reconstitute prior to being drawn into a 6 cc. syringe. Sodiumchloride concentration was adjusted to ensure a final osmolarity of 280mOsm. During preparations, 300 mg of 120 mb silk fibroin was brought upin 3.33 mL of 0.6% Polysorbate-80, 0.317 mL of 200 mg/mL NaCl, and 5.672mL of DI water. Concurrently, 300 mg of 480 mb silk fibroin was broughtup in 3.33 mL 0.6% Polysorbate-80, 0.381 mL of 200 mg/mL NaCl, and 5.675mL DI water. Each individual solution was mixed and incubated at roomtemperature for 30 minutes to dissolve the silk fibroin. The resultingsolutions were stored at 4° C. and aliquoted into 1 cc. syringes priorto injection.

Preparation of Hydrogels

The hydrogel samples were prepared as described below. The lyophilizedsilk fibroin was allowed to fully reconstitute prior to being drawn intoa 6 cc. syringe. During preparation. 300 mg of 120 mb silk fibroin or480 mb silk fibroin were brought up in 3.342 mL 0.6% Polysorbate-80,0.383 mL of 315 mM PB (pH=7.4), and 0.246 mL DI water. Each solution wasmixed and incubated at room temperature for 30 minutes to dissolve thesilk fibroin. The mixtures were aliquoted into 2.13 mL fractions in 3, 6cc, syringes. The samples in Table 21 were named by the process used toprepare and formulate each hydrogel. For example, in the sample named120 mb; hyd; 0% cxbst; 3% SFf; 0% CXBf; 40% PEG4kf, “120 mb” refers tosilk degummed with a 120-minute boil, “hyd” refers to the formulation ofthe sample as a hydrogel, “0% cxbst” refers to a preparation from astock solution of 0% of celecoxib, “3% SFf” refers to a formulation with3% (w/v) silk fibroin, “0% CXBf” refers to a formulation with 0% (w/v)celecoxib, and “40% PEG4kf” refers to a formulation with 40% (w/v)PEG4k. Some samples were prepared with P188 (% P188f). Some samples wereprepared with silk fibroin degummed with a 120-minute boil (120 mb). The120 mb solution control contained 0.2% Polysorbate-80, 22 mM phosphatebuffer, and 6.34 mg/mL NaCl. The 480 mb solution control contained 0.2%Polysorbate-80, 22 mM phosphate buffer, and 6.28 mg/mL NaCl. The 120 mbhydrogel with PEG4k contained 0.2% Polysorbate-80, 22 mM phosphatebuffer, 2.97 mg/mL NaCl, and 15 mM HCl. The 120 mb hydrogel with P188contained 0.2% Polysorbate-80, 22 mM phosphate buffer, and 5.99 mg/mLNaCl. The 480 mb hydrogel with P188 contained 0.2% Polysorbate-80, 22 mMphosphate buffer, and 5.95 mg/mL NaCl.

TABLE 21 Descriptions of samples for in vivo silk fibroin hydrogelexperiments Silk- Silk- Fibroin Fibroin Excipient Ratio Boil Conc. Conc.SF to Sample name Description Time (%) Excipient (%) Excipient PBScontrol PBS — — — — — 120 mb control 120 mb 120 3 — — — Solution 480 mbcontrol 480 mb 480 3 — — — Solution 120 mb; hyd; 3% 120 mb; 120 3 PEG 400.075 0% cxbst; 40% PEG 4 kDa 3% SFf; 4 kDa 0% CXBf; 40% PEG4kf 120 mb;hyd; 3% 120 mb; 120 3 P188 10 0.3 0% cxbst; 10% P188 3% SFf; 0% CXBf;10% P188f 480 mb; hyd; 3% 480 mb; 480 3 P188 10 0.3 0% cxbst; 10% P1883% SFf; 0% CXBf; 10% P188f

Excipient solutions were prepared so that a 0.75:1 mix of silk-fibroinsolution: excipient solution would result in the desired finalformulations. The pH of polyethylene glycol (PEG) hydrogels was adjustedusing hydrochloric acid (from Sigma, St. Louis, Mo.) to account for thechanges in pH observed when mixing phosphate buffer and PEG. Theexcipient solutions were drawn up into a second 6 mL, syringe. Thecorresponding solutions of excipients were prepared as described inTable 22. A 2.87 mL volume of each excipient was aliquoted into asyringe for subsequent mixing with the silk fibroin to generate thedesired formulation. Excipients included NaCl, polyethylene glycol(PEG), and poloxamer 188 (P188). For each sample, the syringe of therepresentative silk fibroin solution was connected to a syringe of itsdesignated excipient solution via a B Braun fluid dispensing connector.The contents of the syringes were then mixed until homogenous. Thehydrogels had an osmolarity of 280 mOsm. The resulting samples wereincubated on a rotator for 24 hours at 37° C. The pH values of thesamples were measured with a glass pH probe, and they were adjusted withhydrochloric acid. The samples had a final (w/v) ratio of silk fibrointo excipient of between 0.01 and 0.5. The samples were then separatedinto 0.4 mL aliquots in I cc syringes, and they were stored at 4° C.until time of injection. Formulations of the hydrogels contained sodiumchloride, 0.2% (w/v) Polysorbate-80, and 22 mM phosphate buffer atpH=7.4 for the P188-containing hydrogels. Some hydrogel formulationscomprised 10% P188 and 10.4 mg/mL sodium chloride with a pH of 7.4.Formulations contained hydrochloric acid, sodium chloride, 0.2% (w/v)Polysorbate-80, and 22 mM phosphate buffer at pH=7.4 for the PEG 4kDa-containing hydrogels. Some formulations contained 40% PEG 4 kDa. 5.2mg/mL sodium chloride, and 22 mM hydrochloric acid, with a pH of 7.4.

TABLE 22 Solution preparations for excipients NaCl mg/mL mL mL mL uL 200needed to stock stock mg/mL uL DI uL 1N Sample in exc. make PEG P188NaCl Water HCl 120 mb; hyd; 0% cxbst; 5.17 4 3.72 0 103 69 108 3% SFf;0% CXBf; 40% PEG4kf 120 mb; hyd; 0% cxbst; 10.43 4 0 3.48 208.6 311 0 3%SFf; 0% CXBf; 10% P188f 480 mb; hyd; 0% cxbst; 10.36 4 0 3.48 207.2 3130 3% SFf; 0% CXBf; 10% P188f

Administration of Hydrogels

The subjects were New Zealand white rabbits with a mass of 3-4 kg. Therabbits were separated into six groups, with three rabbits in eachgroup. Each group was given an intravitreal injection with theformulation as described in Table 23. All injections were performed inthe left eye, with the right eye remaining naïve to serve as anintra-animal control.

TABLE 23 Experimental groups of rabbits for the study of silk fibroinhydrogels Group Description Name of Samples Administered 1 PBS PBScontrol 2 120 mb Solution 120 mb control 3 480 mb Solution 480 mbcontrol 4 3% 120 mb; 40% PEG 4 kDa 120 mb; hyd; 0% cxbst; 3% SFf; 0%CXBf; 40% PEG4kf 5 3% 120 mb; 10% P188 120 mb; hyd; 0% cxbst; 3% SFf; 0%CXBf; 10% P188f 6 3% 480 mb; 10% P188 480 mb; hyd; 0% cxbst; 3% SFf; 0%CXBf; 10% P188f

All silk fibroin hydrogel formulations were pre-loaded into sterile 1 ccsyringes, with 0.4 mL in each syringe. Prior to injection, the syringecap was removed, and a sterile 27-gauge, ½″ needle was attached. Thevolume was adjusted to 0.1 mL, and the formulation was injected into theintravitreal space, 2 mm posterior to the limbus.

All procedures were performed under general anesthesia. Animals weregiven a pre-anesthetic (Xylazine 1.1 mg/kg IM, Buprenorphine HCl 2-6mcg/kg IM). Animals were then anesthetized with ketamine 22 mg/kg IM.Animals were placed on a heating pad, and vitals were monitored. Animalswere put on inhalation anesthesia (Isoflurane at 1.5-2%) with 02supplement.

All rabbits had their peri-ocular fur of the left eye trimmed prior tothe procedure. A wire lid speculum was used to hold the eye open. Theeye was rinsed with balanced salt solution (BSS), followed by a rinsewith 5% ophthalmic betadine. The betadine was applied again, immediatelyprior to the injection and post-injection. All rabbits receivedgentamycin ophthalmic ointment to the operative (left) eye in therecovery area post-procedure.

To administer the hydrogels into the intravitreal space, a lid speculumwas inserted into the rabbit's left ye. The conjunctiva was rinsed withBSS solution. Then, the conjunctival sac was prepped with a 5%ophthalmic betadine solution. The hydrogel was then injected into theintravitreal space using standard the double panel technique describedin the earlier in vivo studies of rods and gels. The formulation wasdelivered to the intravitreal space, and the needle was removed.Following injection, the central retinal artery was examined viaindirect ophthalmoscopy to confirm perfusion and 1-2 drops of betadinesolution were added to the conjunctiva prior to removal of the speculum.

Intraocular Pressure and Biocompatibility after Hydrogel Injection

Immediately following the injection, it was noted that the smaller sizeof the animals used in the study lead to hypoperfusion upon injection of0.1 mL of material. The rabbits had a mass of approximately 3 kg. Thehydrogels injected into animals from groups 4-6 formed well defined,cohesive, spherical depots upon injection. These opaque formulationswere easily visualized. The hydrogels for the experiments on the rabbitsin group 4 (120 mb; hyd; 0% cxbst; 3% SFf; 0% CXBf; 40% PEG4kf) were toodifficult to inject. The injection of this formulation was concluded tonot be feasible without the use of an auto-injector. In addition, thelow molecular weight silk hydrogels, used in the formulations for group6, were less opaque than the formulations with high molecular weightsilk, used on groups 4 and 5.

48 hours after injection, 8 days after injection, and 9 days afterinjection the intraocular pressure was measured with a Tono-Pen (seeTable 24 for results). Anterior penlight exams and posterior dilatedfundus exams were also performed at these times. 48 hours after theinjection, all animals exhibited slight conjunctival irritation. Thisresult was attributed to the betadine solution used during theprocedure. All silk hydrogel formulations, as seen in groups 4-6, wereunchanged. The depots were located at the base of the eye, out of thevisual field, and they were cohesive and opaque. The depots from theformulations used in group 6 (480 mb; hyd: 0/cxbst; 3% SFf; 0% CXBf 10%P188f) were less opaque than those of the other hydrogels. The standarddeviation of the intraocular pressure of the right eye for subjects ingroup 4 (noted by was not calculable because only one animal had the IOPof the right eye measured with a method identical to the rest, renderingn=1 for direct comparisons.

TABLE 24 Intraocular pressure measurements at 48 hour exam with silkfibroin hydrogels Left Eye (Injected) Right Eye (Naïve) Average AverageFold change in IOP Standard IOP Standard IOP Group Sample Name (mmHg)Deviation (mmHg) Deviation (Injected/Naïve) 1 PBS control 11.33 0.58 112.65 1.03 2 120 mb control 11.33 2.08 11.5 0.71 0.99 3 480 mb control10.33 1.53 12 1.41 0.86 4 120 mb; hyd; 0% cxbst; 16.33 3.21 20 0.00*0.82 3% SFf; 0% CXBf; 40% PEG4kf 5 120 mb; hyd; 0% cxbst; 14.67 2.5216.33 4.04 0.90 3% SFf; 0% CXBf; 10% P188f 6 480 mb; hyd; 0% cxbst; 15.52.12 17 2.65 0.91 3% SFf; 0% CXBf; 10% P188f

All silk solutions were determined to be well tolerated via the penlight exam at this timepoint. There were no signs of intraocularinflammation or irritation. Any slight hypoperfusion due to the volumeof the injection had been resolved by this time. Compared to the naïvecontralateral eyes (the right eyes), no elevation in intraocularpressure (IOP) was measured with the Tono-Pen. The fold change in IOPbetween the average IOP of injected (left) eye and the average IOP ofnaïve (right) eye for each group was also calculated. In most cases, theIOP of the left eye was measured to be slightly lower than that of theright eye (the control).

With the exception of the PBS control, i.e., group 1, in all instancesthe fold change in the IOP between the injected and the naïve eye ineach group was less than one, which indicated that all formulations withsilk reduced intraocular pressure. Group 4, where the silk wasformulated with 40% PEG (4 kDa), showed the lowest fold change value,which indicated that this formulation was the most effective in reducingthe intraocular pressure.

8 to 9 days after the injection, all conjunctival irritation hadsubsided. All silk hydrogel formulations (groups 4-6) were mainlyunchanged since the 48 hour examination. The depots were still presentat the base of the eye, out of the visual field, and they were stillcohesive and opaque. The depots from the formulations used in group 6(480 mb; hyd; 0% cxbst; 3% SFf; 0% CXBf; 10% PI88f) were still lessopaque than those of the other hydrogels. The intraocular pressuremeasurements using a Tono-Pen were also made at day 8/9 following thehydrogel injection. The results were shown in Table 25.

TABLE 25 Intraocular pressure measurements at 8 or 9 day exam with silkfibroin hydrogels Left Eye (Injected) Right Eye (Naïve) Average AverageLeft Eye Right Eye Fold change in IOP Standard IOP Standard IOP GroupSample name (mmHg) Deviation (mmHg) Deviation (Injected/Naïve) 1 PBScontrol 8.00 4.00 13.00 3.61 0.62 2 120 mb control 11.67 0.58 13.67 2.080.85 3 480 mb control 13.00 2.65 14.33 2.52 0.91 4 120 mb; hyd; 15.671.53 17.33 4.93 0.90 0% cxbst; 3% SFf; 0% CXBf; 40% PEG4kf 5 120 mb;hyd; 16.00 10.44 10.33 3.79 1.55 0% cxbst; 3% SFf; 0% CXBf; 10% P188f 6480 mb; hyd; 12.00 3.61 24.33 10.41 0.49 0% cxbst; 3% SFf; 0% CXBf; 10%P188f

All hydrogel formulations, silk solutions, and PBS solutions weredetermined to be well tolerated via clinical examination. There were nosigns of intraocular inflammation or irritation. Compared to the naïvecontralateral eyes (the right eyes), no elevation in intraocularpressure (OP) was measured with the Tono-Pen. Animals in groups 1-4 weresacrificed 9 days post-injection. Animals in groups 4-6 were sacrificed8 days post-injection

After 8-9 days, the fold change of the intraocular pressures between theinjected eye and the naïve eye changed more drastically. Almost everygroup experienced a decrease in the fold change, which indicated thatthese formulations with silk reduced intraocular pressure moredrastically over time. Group 6 (480 mb; hyd; 0% cxbst; 3% SFf; 0% CXBf;10% P188f) showed the lowest fold change value, which indicated thatthis formulation was the most effective in reducing the intraocularpressure. Meanwhile, group 5 (120 mb; hyd; 0% cxbst; 3% SFf; 0% CXBf;101% P188f), the hydrogels of which were formulated with a highermolecular weight silk fibroin, experienced an increase in the foldchange, which indicated that this formulation increased intraocularpressure over time.

Example 13. In Vivo Study of Silk Fibroin Hydrogels with Celecoxib in anAnimal Model

As seen in the studies of silk fibroin hydrogels formulated without atherapeutic agent, all buffers and stock solutions were prepared understerile conditions unless otherwise indicated. All formulations wereprepared with SOHO silk yam. The poloxamer-188, sodium chloride, andhydrochloric acid were from Sigma-Aldrich (St. Louis, Mo.), the PEG4 kDawas from Clariant, Charlotte N.C., and the celecoxib (CXB) was fromCipla, Miami Fla. Polysorbate-80 was purchased from Croda (Snaith UK).Potassium phosphate monobasic and potassium phosphate dibasic werepurchased from Sigma Aldrich Fine Chemical (SAFC, St. Louis Mo.).Phosphate buffered saline was purchased from Gibco (USA). Multiplepreparations of the same formulations were used.

Preparation of Celecoxib Experimental Controls

All controls were prepared as described for the in vivo experiments ofsilk fibroin hydrogels with no therapeutic agent. Briefly, a 27.8%suspension of celecoxib (CXB) was prepared from 4.15 g dry heat treated(DHT) CXB in 10.78 mL of 0.79% Polysorbate-80 and mixed untilhomogenous. 1.789 mL of the 27.8% CXB suspension was diluted to 5 mL viathe addition of 0.349 mL 315 mM PB (pH=7.4), 0.158 mL of 200 mg/mL NaCl,and DI water. The resulting 10% CXB suspension was immediately aliquotedinto 0.4 mL fractions in 1 cc syringes so that it remained homogenous,and the fractions were stored on ice until injection.

Preparation of Silk Fibroin Hydrogels with 10% Celecoxib

The hydrogel samples were prepared as described in the experiments onhydrogels without a therapeutic agent. Hydrogels were prepared from bothhigh molecular weight (120 mb) and low molecular weight (480 mb) silkfibroin. 300 mg of either 120 mb or 480 mb silk fibroin were brought upin 3.589 mL of the 27.8% CXB suspension and 0.381 mL of 315 mM PB(pH=7.4). The resulting solutions were incubated at room temperature andmixed for 30 minutes until homogenous. The silk fibroin solutions werethen aliquoted into 2.13 mL fractions in 5 cc syringes. The samples inTable 26 are named by the process used to prepare and formulate eachhydrogel. For example, in the sample named 120 mb; hyd; 27.8% cxbst; 3%SFf; 10% CXBf; 10% P188f. “120 mb” refers to silk degummed with a120-minute boil, “hyd” refers to the formulation of the sample as ahydrogel, “27.8% cxbst” refers to a preparation from a stock solution of27.8% of celecoxib, “3% SFf” refers to a formulation with 3% (w/v) silkfibroin, “10% CXBf” refers to a formulation with 10% (w/v) celecoxib,and “10% P188f” refers to a formulation with 10% (w/v) poloxamer 188.Some samples were prepared with silk fibroin degummed with a 120-minuteboil (120 mb). The 10% CXB suspension contained 0.2% Tween-80, 22 mMphosphate buffer, and 6.32 mg/mL NaCl. The 120 mb hydrogel contained0.2% Tween-80, 22 mM phosphate buffer, and 5.99 mg/mL NaCl. The 480 mbhydrogel contained 0.2% Tween-80, 22 mM phosphate buffer, and 5.95 mg/mLNaCl.

TABLE 26 Descriptions of samples for in vivo experiments with 3% (w/v)silk fibroin (SF) hydrogels formulated with 10% (w/v) celecoxib and 10%P188 Ratio Ratio SF SF P188 SF to Ratio CXB to Sample Boil Conc. Conc.Excipient CXB Excipient Name Description Time (%) (%) (P188) to SF(P188) CXB:SF:P188 10% CXB 10% CXB — — — — — — — control Suspension 120mb; 3% 120 mb; 120 3 10 0.3 3.33 1 10:3:10 hyd; 10% P188; 27.8% cxbst;10% CXB 3% SFf; 10% CXBf; 10% P188f 480 mb; 3% 480 mb; 480 3 10 0.3 3.331 10:3:10 hyd; 10% P188; 27.8% cxbst; 10% CXB 3% SFf; 10% CXBf; 10%P188f

The corresponding solutions of excipients were prepared as described inTable 27. As with the hydrogels without CXB, a 2.87 mL volume of eachexcipient was aliquoted into a 3 cc syringe for subsequent mixing withthe silk fibroin to generate the described formulation. For each sample,the syringe of the representative silk fibroin solution was connected toa syringe of its designated excipient solution via a B Braun fluiddispensing connector. The contents of the syringes were then mixed untilhomogenous. The resulting samples were incubated on a rotator for 24hours at 37° C. and then separated into 0.4 mL aliquots in cc syringes.The pH values of the samples were measured with a glass pH probe, andthey were adjusted with hydrochloric acid. The resulting hydrogels hadratio of silk fibroin to excipient to between 0.01 and 0.5, a ratio ofcelecoxib to silk fibroin of between 0.1 and 5, and a ratio of celecoxibto excipient of 1. The ratio of celecoxib to silk fibroin to excipientwas 10:3:10. Formulations of the hydrogels contained sodium chloride,0.2% (w/v) Polysorbate-80, and 22 mM phosphate buffer at pH=7.4 for theP188-containing hydrogels. Some formulations comprised 10% P188, 10%CXB, and 10.4 mg/mL sodium chloride at a pH of 7.4.

TABLE 27 Excipient solution preparations for hydrogels with celecoxib.NaCl mg/mL mL mL μL 200 μL needed to stock mg/mL DI Sample Name in exc.make P188 NaCl Water 120 mb; hyd; 10.44 4 3.48 208.8 311.2 27.8% cxbst;3% SFf; 10% CXBf; 10% P188f 480 mb; hyd; 10.36 4 3.48 207.2 312.8 27.8%cxbst; 3% SFf; 10% CXBf; 10% P188f

Administration of Hydrogels

The methods of administration of silk fibroin hydrogels with celecoxibwere identical to those used to administer the hydrogels withoutcelecoxib. Briefly, the subjects were New Zealand white rabbits with amass of 4 kg. The rabbits were separated into three groups, with threerabbits in each group. Each group was injected with the formulation asdescribed in Table 28. All injections were performed in the left eye,with the right eye remaining naïve to serve as an intra-animal control.

TABLE 28 Experimental groups of rabbits for the study of silk fibroinhydrogels formulated with celecoxib. Name of Sample Group DescriptionAdministered 1 10% CXB Suspension 10% CXB control 2 3% HMW (120 mb)Silk; 120 mb; hyd; 27.8% cxbst; 10% Poloxamer-188; 3% SFf; 10% CXBf; 10%10% CXB P188f 3 3% LMW (480 mb) Silk; 480 mb; hyd; 27.8% cxbst; 10%Poloxamer-188; 3% SFf; 10% CXBf; 10% 10% CXB P188f

All silk fibroin hydrogel formulations were pre-loaded into sterile 1 ccsyringes, with 0.4 mL in each syringe. Prior to injection, the syringecap was removed, and a sterile 27-gauge, ½″ needle was attached. Thevolume was adjusted to 0.1 mL, and the formulation was injected into theintravitreal space, 2 mm posterior to the limbus. The method ofinjection was as described for the in vivo studies of silk fibroinhydrogels without celecoxib. Briefly, a lid speculum was inserted intothe rabbit's left eye. The conjunctiva was rinsed with BSS solution.Then, the conjunctival sac was prepped with a 5% ophthalmic betadinesolution. The hydrogel was then injected into the intravitreal spaceusing the double panel technique described in the earlier in vivostudies of rods and gels. The formulation was delivered, and the needlewas removed. Following injection, the central retinal artery wasexamined via indirect ophthalmoscopy to confirm perfusion and 1-2 dropsof betadine solution were added to the conjunctiva prior to removal ofthe speculum.

All procedures were performed under general anesthesia. All rabbits hadtheir peri-ocular fur of the left eye trimmed prior to the procedure.All rabbits received gentamycin ophthalmic ointment to the operative(left) eye in the recovery area post-procedure.

Intraocular Pressure and Biocompatibility after Injection of Hydrogelswith Celecoxib

24 hours after the injection, and 7 days after the injection, theintraocular pressure was measured with a Tono-Pen, as shown in Table 29.Anterior penlight exams and posterior dilated fundus exams were alsoperformed at these times. Even though larger animals, with a mass ofapproximately 4 kg, were used for this study than those used for thestudy of hydrogels without therapeutics, it was noted that hypoperfusionstill occurred upon injection of 0.1 mL. This was expected as thisvolume was likely the largest volume that could be well-tolerated.Animal CCN-23 only received a half-volume injection and was thereforeconsidered not usable for the current study. However, the injection didseem well-tolerated, and may be a suitable volume for injection infuture studies. All hydrogel groups were more difficult to inject thantheir corresponding controls without drug. The hydrogels formed welldefined, cohesive depots upon injection. These opaque formulations wereeasily visualized. Furthermore, the suspension, rather than immediatelydispersing, stayed together well in the vitreous space.

TABLE 29 Intraocular pressure measurements at 24 hour exam with silkfibroin hydrogels with celecoxib Left Eye (Injected) Right Eye (Naïve)Average Average Left Eye Right Eye Fold change in IOP Standard IOPStandard IOP Group Sample Name (mmHg) Deviation (mmHg) Deviation(Injected/Naïve) 1 10% CXB control 10.00 4.58 11.33 1.53 0.88 2 120 mb;hyd; 8.00 2.65 13.33 4.16 0.60 27.8% cxbst; 3% SFf; 10% CXBf; 10% P188f3 480 mb; hyd; 7.67 0.58 13.00 1.00 0.59 27.8% cxbst; 3% SFf; 10% CXBf;10% P188f

24 hours after the injection, all animals exhibited slight conjunctivalirritation. This result was attributed to the betadine solution usedduring the procedure. All silk hydrogel formulations, as well as thesuspension, were physically unchanged. All formulations were determinedto be well-tolerated via ocular examination. There were no observedsigns of intraocular inflammation or irritation. Any slighthypoperfusion due to injection had resolved. Compared to the naïvecontralateral eyes (the right eyes), no elevation in intraocularpressure (IOP) was measured with the Tono-Pen. In most cases, the IOP ofthe injected left eye was measured to be lower than that of the righteye (the control). The fold change of the intraocular pressure betweenthe injected eye and the naïve eye decreased for all silk fibroinformulations relative to the CXB suspension control.

The eyes were examined again during a 7-day exam. The intraocularpressure was also measured at this timepoint, seen in Table 30A.

TABLE 30A Intraocular pressure measurements at 7-day exam with silkfibroin hydrogels with celecoxib Left Eye (Injected) Right Eye (Naïve)Average Average Left Eye Right Eye Fold change in IOP Standard IOPStandard IOP Group Sample Name (mmHg) Deviation (mmHg) Deviation(Injected/Naïve) 1 10% CXB control 6.67 0.58 13.33 0.58 0.50 2 120 mb;hyd; 7.67 2.31 10.00 4.36 0.77 27.8% cxbst; 3% SFf; 10% CXBf; 10% P188f3 480 mb; hyd; 8.67 4.04 11.67 2.89 0.74 27.8% cxbst; 3% SFf; 10% CXBf;10% P188f

By the 7-day examination, all conjunctival irritation had subsided. Thematerials were concluded to be tolerated at 7 days. There were noobvious signs of inflammation. The hydrogels and the suspensions werecohesive at the 7-day timepoint. No elevation was detected inintraocular pressure compared to the naïve contralateral eyes. There wasa slight trend toward lower intraocular pressures in the CXB-treatedeyes. The fold change in the IOP between the injected and the naïve eyein each group was less than one, which indicated that all formulationsreduced intraocular pressure. The fold change also revealed that theformulations with silk reduced the intraocular pressure to a lesserextent than the CXB suspension.

The analysis of the intraocular pressure was continued, as seen in Table30B. At 4.5 months after administration, the CXB-containing hydrogelsshowed a slight decrease in intraocular pressure, similar to that of theCXB suspension. In addition, the intraocular pressure was measured to bethe same as the untreated eye at 7 months after administration ofhydrogel with no CXB. No local inflammation, hemorrhage, or othercomplications were detected 7 months after administration.

TABLE 30B Continued measurements of intraocular pressure of silk fibroinhydrogels with and without celecoxib Left Eye (Injected) Right Eye(Naïve) Average Average Left Eye Right Eye Fold change in IOP StandardIOP Standard IOP Sample Name Days (mmHg) Deviation (mmHg) Deviation(Injected/Naïve) 10% CXB 127 7.00 2.58 9.25 2.22 0.76 control 480 mb;hyd; 127 6.00 2.16 7.75 3.30 0.77 27.8% ccxbst; 3% SFf; 10% CXBf; 10%P188f 480 mb; hyd; 197 10.33 3.21 10.67 3.21 0.97 27.8% cxbst; 3% SFf;0% CXBf; 10% P188f

Example 14. Histopathology Studies of Rabbit Eyes with Hydrogels

Following the experiments on intraocular pressure and biocompatibility,the animals were sacrificed, and both eyes were immediately enucleatedand placed into a solution of 10% formalin. After 24 hours, the eyeswere transferred to a solution of 70% ethanol for subsequenthistopathology studies. Thirty-two rabbit eyes were submitted for thestudy. The eyes were processed into two blocks per sample. One slide perblock was sectioned and stained with hematoxylin and eosin (H&E). Theglass slides were evaluated by a board-certified veterinary pathologistvia light microscopy. Histologic legions were graded for severity(0=absent; 1=minimal; 2=mild; 3=moderate; 4=marked; 5=severe).

Histologic findings in this study consisted of an infiltration of mixedinflammatory cells into the vitreous chamber, including heterophils(neutrophils), lymphocytes, plasma cells, macrophages and raremultinucleated giant cells. Inflammatory cells were primarily present inthe region of the or a ciliaris retinae and variably surrounded presumedinjected material within the vitreous chamber. This material ranged frombasophilic flocculent to granular material, to more discrete,non-staining slightly refractile material less than 10 μm in diameter,to non-staining cleft-shaped material (resembling cholesterol clefts).Similar inflammatory cells infrequently extended into the adjacentciliary body epithelium or retina. A granuloma, characterized byaggregation of macrophages and multinucleated giant cells, surroundingnon-staining cholesterol cleft-like material and phagocytized debris,was present in the conjunctiva of one animal. Mononuclear inflammatoryinfiltrate was characterized by infiltration or aggregation oflymphocytes and plasma cells, with rare heterophils, in the conjunctiva.Infiltration of similar mononuclear cells into the iris was observed inone animal. Elevation of the retina from the retinal pigmentedepithelium, present in many samples, was not associated with otherfeatures supportive of true retinal separation and this finding wastherefore considered an artifact.

Means of the grades of the histologic lesions were examined, as well asthe standard error of the mean (SEM), shown in Table 31. Mean scores formixed inflammatory cell infiltration into the vitreous chamber were onlyobserved in samples with intravitreal injections containing 10%celecouib (CXB) (Groups 8-10). The highest mean score was observed inthe 10% CXB suspension alone group (Group 8). The animal withconjunctival granuloma was also in this group. Mean scores forconjunctival mononuclear cell infiltration severity were similar amongall groups, regardless of injection status or injection material. Focaliris infiltration of inflammatory cells was only present in one animal,which had been from the low molecular weight (MW) solution group.

TABLE 31 H&E grades of the rabbit eye histopathology data of animalstreated with silk fibroin compositions (Standard error of the mean)Inflammation, mixed, Infiltrate, Infiltrate, Name of injected vitreousmononuclear, Granuloma, mononuclear, Group sample chamber irisconjunctiva conjunctiva Group 1 Untreated 0 (±0.00) 0 (±0.00) 0 (±0.00)0.50 (±0.50) (Untreated) Group 2 (PBS) PBS 0 (±0.00) 0 (±0.00) 0 (±0.00)0.33 (±0.33) Group 3 (HMW 120 mb control 0 (±0.00) 0 (±0.00) 0 (±0.00)0.67 (±0.33) Solution) Group 4 (LMW 480 mb control 0 (±0.00) 0.33(±0.33)   0 (±0.00)   0 (±0.00) Solution) Group 5 (3% 120 mb; hyd; 0%cxbst; 0 (±0.00) 0 (±0.00) 0 (±0.00)   1 (±0.00) HMW SF; 40% 3% SFf; 0%CXBf; 4 kDa PEG) 40% PEG4kf Group 6 (3% 120 mb; hyd; 0% cxbst; 0 (±0.00)0 (±0.00) 0 (±0.00) 1.33 (±0.33) HMW SF, 10% 3% SFf; 0% CXBf; P188) 10%P188f Group 7 (3% 480 mb; hyd; 0% cxbst; 0 (±0.00) 0 (±0.00) 0 (±0.00)  1 (±0.00) LMW SF, 10% 3% SFf; 0% CXBf; P188) 10% P188f Group 8 (10%10% CXB control 2 (±0.00) 0 (±0.00) 0.67 (±0.67)     1 (±0.00) CXBSuspension) Group 9 (3% 120 mb; hyd; 0.75 (±0.48)   0 (±0.00) 0 (±0.00)0.75 (±0.25) HMW SF, 10% 27.8% cxbst; 3% SFf; P188, 10% CXB 10% CXBf;10% P188f Suspension) Group 10 (3% 480 mb; hyd; 0.67 (±0.33)   0 (±0.00)0 (±0.00) 0.67 (±0.33) LMW SF, 10% 27.8% cxbst; 3% SFf; P188, 10% CXB10% CXBf; 10% P188f Suspension)

Imaging of an untreated eye displayed no lesions at the or a ciliarisretinae. The normal vitreous humor was visible as an acellular, slightlyeosinophilic wispy material in the vitreous chamber. The ciliary body,retina, and sclera were also visible in the images. Imaging of an eyetreated with a 10%4,CXB suspension demonstrated inflammatoryinfiltration into the vitreous chamber. There were more abundantheterophils, lymphocytes, and macrophages. Inflammatory cells were alsorarely present in the retina.

Imaging of an eye treated with 120 mb; hyd; 0% cxbst; 3% SFf; 0% CXBf;10% P188f showed that there was a mild infiltration of lymphocytes andmononuclear plasma cells within the conjunctiva.

Imaging of an eye treated with an intravitreal injection of 120 mb; hyd;27.8° % cxbst; 3% SFf; 10% CXBf; 10% P188f demonstrated that theinjected vitreous material was more basophilic and granular compared tothe normal vitreous humor. Macrophages, and fewer lymphocytes andheterophils, surrounded and infiltrated this material.

The major finding associated with intravitreal injections in this studywas vitreous chamber mixed inflammation, limited to the eyes receivinginjections containing 10% CXB. Mixed inflammatory cell infiltration inthe vitreous chamber was only observed in groups receiving 10% CXB, witha 3-fold increase in the scores in the 10% CXB suspension group comparedto groups 9 and 10 where the CXB was formulated with silk. This resultshowed that CXB silk formulations can potentially reduce theinflammatory responses seen with CXB only injections.

The observed inflammation was likely due to the presence of CXB. It ispossible that the inflammation is a result of slight toxicity due tohigh initial levels of CXB in the vitreous. In the silk fibroinformulations, the initial levels of CXB in the vitreous were lowerlikely due to the slower release of the therapeutic agent. Theinflammation might also have been caused by the suspension form of CXB.The smaller particles could induce a macrophage response; they could beengulfed by macrophages and ultimately lead to inflammation. Bycontrast, the hydrogel would contain these particles and reduce theresulting inflammation.

In most groups, there was minimal to mild conjunctival mononuclearinfiltration. This inflammatory infiltrate typically targetedpresumptive injected material, with a range in inflammatory responsefrom primarily acute (heterophilic/neutrophilic) to a more foreignbody-type reaction with more numerous macrophages ingesting the injectedmaterial. Extension of inflammatory cells into the surrounding tissueswas infrequently present and was not associated with ciliary epithclialor retinal degeneration. The granuloma present in the conjunctiva of oneeye (10% CXB Suspension group) was considered secondary to the injectionprocedure. Conjunctival and iridal mononuclear inflammatory cellinfiltration was present in numerous eyes from both untreated andtreated groups; these findings were considered background lesions thatwere unrelated to treatment. The retinal tissue was considered normal.

Additional histopathology studies were performed on animals sacrificed 6and 7 months after administration of the silk fibroin hydrogels (480 mb;hyd; 0% cxbst; 3% SFf; 0% CXBf; 10% P188f). At 6 and 7 months afteradministration, the injected material was free of cellular infiltrate.No other histologic findings were observed. No local inflammation,hemorrhage, or other complications were observed. Ultimately thehydrogels were determined to be biocompatible and well-tolerated in theintravitreal space for at least 7 months after administration.

Example 15. Histopathology Studies of Rabbit Eyes with Silk Rods

Following the experiments on intraocular pressure and biocompatibility,the animals were sacrificed, and both eyes were immediately enucleatedand placed into a solution of 10% formalin. After 24 hours, the eyeswere transferred to a solution of 70% ethanol for shipment andsubsequent histopathology studies. The eyes were from animals sacrificed1 week after administration of the silk rods. Four formalin-fixed rabbiteyes were processed into two blocks per sample. One slide per block wassectioned and stained with hematoxylin and eosin (H&E). The glass slideswere evaluated by a board-certified veterinary pathologist, using lightmicroscopy. Histologic lesions were graded for severity (0=absent;1=minimal; 2=mild; 3=moderate; 4=marked; 5=severe), as seen in Table 32.L denoted the left eye, while R denoted the right eye.

TABLE 32 H&E grades of the rabbit eye histopathology data of animalstreated with silk fibroin rod compositions; P = present, NP = notpresent H&E Mixed Foreign infiltrate, material, vitreous vitreouschamber chamber (surrounding Mixed Sample (presumptive foreignDegeneration, inflammation, Treatment Name Eye Block rod) material) lensfiber conjunctiva Silk- 480 mb; 56L 1 NP 0 0 0 Fibroin/ 0.5 mm; 2 P 1 00 CXB Rod 40% st; 58L 1 P 1 0 2 100 mgsf; 2 P 0 0 1 200 mgcxb; lyo; 59L1 NP 0 2 0 33.3% sf; 2 NP 0 0 0 66.7% cxb Untreated — 56R 1 NP 0 0 0 2NP 0 0 0

Foreign material, presumably the injected celecoxib (CXB) rod, waspresent in the vitreous chamber of two eyes, near the or a ciliarisretinae. This material was a solid mass of amphophilic material,approximately 500 μm in diameter, containing non-staining clefts andvacuoles. This material was variably loosely surrounded or minimallyinfiltrated by low numbers of macrophages, rare heterophils and scanthemorrhage. Inflammation was not observed in other areas of the vitreouschamber or within the adjacent ciliary body/uveal tract or retina. Inone eye, slight lens fiber degeneration was present. This finding mightbe associated with the injection procedure. Mixed inflammatory cellinfiltration was observed in the conjunctiva from one eye. This findingwas determined to be a background lesion, and it was unlikely to beassociated with test article administration. Ultimately, histopathologicevaluation revealed minimal infiltration of low numbers of macrophagesand rare heterophils. No other inflammation of note within the vitreouscavity, adjacent ciliary body, or retina, was detected. The silk rodswere well tolerated in the intravitreal space.

Additional histopathology studies were performed on animals sacrificed 4months after administration of the silk fibroin rods. The studiesdetermined 2 out of the 3 rods to be acellular with visible implant. In1 of the 3 rods the implant was surrounded and infiltrated bylymphocytes, macrophages, and multinucleated giant cells. Most of thesamples did not illicit a significant inflammatory response. Ultimatelythe rods were determined to be biocompatible and well-tolerated in theintravitreal space for at least 4 months after administration.

Example 16. Release of Protein Cargo and Relation of Release Kinetics toProtein Molecular Weight in Silk Fibroin Rods

Silk fibroin rods were prepared from silk fibroin degummed with a 480 mbor a 120 mb. Sodium chloride was purchased from Chemsavers (BluefieldVa.). Polysorbate-80 was purchased from Croda (Snaith, United Kingdom).Phosphate buffered saline (10×PBS) was purchased from Gibco (USA).Sodium phosphate dibasic, sodium phosphate monobasic, human lysozyme,sucrose, Bovine Serum Albumin (BSA), trehalose, and poloxamer-188 (P188)were purchased from Sigma-Aldrich (St. Louis, Mo.). Sodium azide andglycerol were purchased from Fisher Chemical (Waltham, Mass.).Bevacizumab was purchased from Genentech Inc. (San Francisco, Calif.).Human immunoglobulin G (IgG) was purchased from Innovative Research(Novi, Mich.).

Preparation of Silk Fibroin Rods with Proteins

Silk fibroin rods were formulated with proteins, and the controlledrelease of said proteins were monitored in vitro. Silk fibroin rods wereformulated with lysozyme (molecular weight=14 kDa; Sigma-Aldrich, St.Louis, Mo.), bovine serum albumin (BSA) (molecular weight=67 kDa;Sigma-Aldrich, St. Louis, Mo.), bevacizumab (molecular weight=150 kDa;Genentech Inc., San Francisco, Calif.), and Immunoglobulin G (IgG) asdescribed in Table 33. The aqueous processing of the silk fibroin rodswas amenable to aseptic conditions. Some of the silk fibroin rods were5% (w/w) of the respective protein. The silk fibroin rods are named bythe process used to prepare and formulate each rod. For example, the rodnamed “480 mb; 1 mm; 5% bevst; lyo; 75% sf; 3% bevacizumab; 22% sucrose”refers to rod prepared from silk degummed with a 480-minute boil (480mb), a 1 mm diameter (1 mm), prepared from a 5% w/v bevacizumab stocksolution (bevst), lyophilization (lyo), a theoretical w/w percentage of75% silk fibroin (75% sf) a theoretical w/w percentage of 3% bevacizumab(3% bevacizumab), and a theoretical w/w percentage of 22% sucrose (22%sucrose). Other potential components of the rods described in the nameincluded gelation at 4° C. (4° C.), a preparation from a stock solutionof silk fibroin (e.g. 40% st), a theoretical w/w percentage of IgG (°igg), a theoretical w/w percentage of lysozyme (% lysozyme) apreparation from silk fibroin degummed with a 120-minute boil (120 mb),preparation from silk fibroin degummed with 90-minute boil (90 mb),atheoretical w/w percentage of bovine serum albumin (% bsa), and atheoretical w/w percentage of trehalose (% trehalose). Sample 205-1contained 133.3 mM phosphate buffer. 205-2 contained 133.3 mM phosphatebuffer. 205-5 contained 133.3 mM phosphate buffer. Rods with bevacizumabalso contained small amounts of the buffer that the product was providedin (trehalose, a sodium phosphate buffer, and polysorbate-20).

TABLE 33 Silk rods formulated with proteins Sample Time Silk- mass of offibroin Protein Excipient each Sample heating Conc. conc. Conc.replicate No. Sample Name (mb) % Protein % Excipient % (mg) 204-05 480mb; 1 mm; 480 75 Bevacizumab 3 Sucrose 22 10.07 5% bevst; lyo; 9.82 75%sf; 9.74 3% bevacizumab; 2.2% sucrose 205-01 480 mb; 1 mm; 480 85Bevacizumab 5 Sucrose 10 9.71 5% bevst; lyo; 10.17 85% sf; 10.05 5%bevacizumab; 10% sucrose 205-02 480 mb; 1 mm; 480 73 Bevacizumab 5Sucrose 22 10.6 5% bevst; lyo; 9.87 73% sf; 10.36 5% bevacizumab; 22%sucrose 202-03 480 mb; 1 mm; 480 95 Bevacizumab 5 — — 6.18 30% st; 5%bevst; 7.16 lyo; 95% sf; 6.93 5% bevacizumab 205-04 480 mb; 1 mm; 480 85IgG 5 Sucrose 10 7.64 40% st; 4° C.; lyo; 8.3 85% sf; 5% igg; 7.7 10%sucrose 205-05 480 mb; 1 mm; 480 95 Lysozyme 5 — — 6.68 lyo; 95% sf; 7.85% lysozyme 6.22 205-06 480 mb; 1 mm; 480 85 Lysozyme 5 Sucrose 10 8.66lyo; 85% sf; 7.94 5% lysozyme; 9.23 10% sucrose 205-07 480 mb; 1 mm; 48075 Lysozyme 25 — — 8.3 lyo; 75% sf; 9.64 25% lysozyme — 205-08 480 mb; 1mm; 480 65 Lysozy me 25 Sucrose 10 10.4 lyo; 65% sf; 10.02 25% lysozyme;7.98 10% sucrose 205-A 120 mb; 1 mm; 120 95 Lysozyme 5 — — 6.36 lyo; 95%sf; 5.99 5% lysozyme 5.58 197-09 480 mb; 1 mm; 480 96.5 BSA 2.5Trehalose  1 9.24 40% st; lyo; 8.27 96.5% sf; 8.11 2.5% bsa; 1%trehalose 197-11 120 mb; 1 mm; 120 96.5 13 SA 2.5 Trehalose  1 4.89 30%st; lyo; 4.89 96.5% sf; 5.29 2.5% bsa; 1% trehalose 197-12 120 mb; 1 mm;120 94 BSA 5 Trehalose  1 6.59 30% st; lyo; 6 94% sf; 5% bsa; 6.02 1%trehalose 201-04 480 mb; 1 mm; 480 95 Bevacizumab 5 — — 8.45 5% bevst;4° C.; 8 lyo; 95% sf; 7.89 5% bevacizumab 209-05 90 mb; 1 mm; 90 97.5IgG 2.5 — — — 30% st; 4° C.; lyo; — 97.5% sf; — 2.5% igg 209-A 480 mb; 1mm; 480 85 IgG 5 Sucrose 10 — 40% st; 4° C.; lyo; — 85% sf; 5% igg; —10% sucrose 191-01 480 mb; 1 mm; 480 94 BSA 5 Trehalose  1 — 40% st;lyo; — 94% sf; 5% bsa; — 1% trehalose 191-02 480 mb; 1 mm; 480 96.5 BSA2.5 Trehalose  1 — 40% st; lyo; — 96.5% sf; — 2.5% bsa; 1% trehalose

To prepare the silk fibroin rods with lysozyme, silk fibroin wasdissolved in lysozyme stock solution to reach the final desiredsilk/lsozyme concentrations. Sucrose (Sigma Aldrich, St. Louis Mo.) wasdissolved in this solution when necessary. Formulations were injectedinto 1.0 mm diameter PTFE tubing. The tubing was capped with Paraflim®and allowed to gel at 37° C. overnight. Once gelling was achieved, thetubing was frozen and lyophilized.

To prepare the silk fibroin rods with BSA, silk fibroin wasreconstituted in sufficient deionized water to reach a finalconcentration of 30 or 40% (w/v). BSA solutions were prepared, from astock solution of 40 mg/mL BSA, with or without trehalose (SigmaAldrich, St. Louis Mo.) and/or polysorbate-80 (Sigma Aldrich, St. LouisMo.). Solutions were mixed between two syringes and extruded into 1.0 mminner diameter PTFE tubing (Grainger, Ill., USA). The tubing was cappedwith Parafilm® and allowed to gel at 4° C. overnight. Once gelling wasachieved, the tubes were frozen and lyophilized. Samples 191-01 and191-02 had 0.1% Tween-80 in the final formulation.

To prepare the silk fibroin rods with bevacizumab, silk fibroin wasreconstituted in sufficient deionized water to reach a finalconcentration of 30% (Sample 202-03) or 40% (remaining samples) (w/v).The reconstituted fibroin was added to a concentrated solution ofbevacizumab (50 mg/mL) to achieve the desired final ratio ofbevacizumab:silk. Rods containing sucrose were prepared from silkfibroin lyophilized with sucrose. Solutions were mixed using two linkedsyringes and then injected into 1.0 mm diameter PTFE tubing. The rodswere capped with Parafilm® and allowed to gel at 4° C. (Sample 201-4only) or 37° C. overnight. Once gelling was achieved, the tubes werelyophilized overnight.

To prepare silk fibroin rods with immunoglobulin G (IgG), silk fibroindegummed with a 480 mb or a 90 mb, was reconstituted in sufficientdeionized water to reach a final concentration of 30 or 40% (w/v). Rodscontaining sucrose were prepared from silk-fibroin lyophilized withsucrose as an additive. Solutions were mixed between two syringes andinjected into 1.0 mm diameter PTFE tubing. The rods were capped withParafilm® and allowed to gel at 4° C. overnight. Once gelling wasachieved, the tubes were frozen and lyophilized.

In Vitro Release Profile of Silk Fibroin Rods Formulated with ProteinAPIs

Silk fibroin rods were cut into 1 cm sections and two sections wereplaced, in triplicate, into 4 mL glass vials. 1 mL of release media(PBS, 0.01% polysorbate-80 (PS80), 0.05% sodium azide) was added to eachvial. Samples were incubated with gentle shaking at 37° C. At 2 hours,1, 2, 3, 7, 10, 14, 21, and 28 days, 100 μL of release media was removedand replaced with 100 μL of fresh release media. Total protein releasedwas quantified via size-exclusion chromatography (SEC) using a WatersX-Bridge Protein BEH SEC, 200 Å, 3.5 μm column. An isocratic flow ofmobile phase (100 mM sodium phosphate (Sigma Aldrich. St. Louis Mo.),200 mM NaCl (Chemsavers, Bluefield Va.) pH 6.8) was run at 0.80 mL/minto elute protein. Protein elution was monitored at 280 and 214 nm usingan Agilent 1290 HPLC system with a photodiode array (PDA) detector.Cumulative percentage of protein released was calculated usingtheoretical loading of the silk fibroin rods.

The average cumulative release percentage of each protein was monitoredover time, as seen in Table 34A and Table 34B. The data suggested thatrelease was related to size-dependent diffusion through the silk fibroinmatrix. The release kinetics and the cumulative release percentagesdecreased with increased molecular weight of the protein to be released.Silk fibroin rods formulated with lysozyme had the highest initial burstpercentage, while rods formulated with bevacizumab had the lowestinitial burst percentage. The initial burst percentages ranged from1-85% over the first 24 hours of the experiment. The cumulative releasepercentage of protein released from each rod were measured intriplicate, except for the specific measurements marked with “*”, whichwere measured in singlicate. Sample 205-07 and sample 197-12, markedwith “***”, were tested in duplicate.

TABLE 34A In vitro release of proteins from silk-fibroin rods;cumulative percentage (%) of API released Sample Days No. Sample Name 00.083 1 2 3 7 204-05 480 mb; 1 mm; 5% bevst; lyo; 75% sf; 0.0 0.5 0.7 —— — 3% bevacizumab; 22% sucrose 205-01 480 mb; 1 mm; 5% bevst; lyo; 85%sf; 0.0 6.6 6.9 — — — 5% bevacizumab; 10% sucrose 205-02 480 mb; 1 mm;5% bevst; lyo; 73% sf; 0.0 2.2 2.7 — — — 5% bevacizumab; 22% sucrose202-03 480 mb; 1 mm; 30% st; 5% bevst; lyo; 0.0 3.2 — — — — 95% sf; 5%bevacizumab 205-04 480 mb; 1 mm; 40% st; 4° C.; lyo; 0.0 5.7 19.4 20.2 —— 85% sf; 5% igg; 10% sucrose 205-05 480 mb; 1 mm; lyo; 95% sf; 0.0 18.727.2 32.4 7.2  7.6 5% lysozyme 205-06 480 mb; 1 mm; lyo; 85% sf; 0.019.4 29.4 34.8 *29.5 10.3 5% lysozyme; 10% sucrose 205-07*** 480 mb; 1mm; lyo; 75% sf; 0.0 17.5 22.4 35.0 37.1 39.7 25% lysozyme 205-08 480mb; 1 mm; lyo; 65% sf; 0.0 48.7 75.0 83.3 71.1 73.9 25% lysozyme; 10%sucrose 205-A 120 mb; 1 mm; lyo; 95% sf; 0.0 11.6 12.8 14.2 10.3 — 5%lysozyme 197-09 480 mb; 1 mm; 40% st; lyo; 96.5% sf; 0.0 0.0 0.0 0.0 0.0— 2.5% bsa; 1% trehalose 197-11 120 mb; 1 mm; 30% st; lyo; 96.5% sf; 0.013.7 21.3 21.6 26.1 — 2.5% bsa; 1% trehalose 197-12*** 120 mb; 1 mm; 30%st; lyo; 94% sf; 0.0 9.1 14.2 16.0 17.9 — 5% bsa; 1% trehalose 201-04480 mb; 1 mm; 5% bevst; 4° C.; lyo; 0.0 56.0 84.5 73.8 58.3 66.8 95% sf;5% bevacizumab

TABLE 34B Standard deviations (%) of the cumulative percentage of APIreleased for the in vitro release of proteins from silk rods Sample DayNo. Sample Name 0 0.083 1 2 3 7 204-05 480 mb; 1 mm; 5% bevst; lyo; 0.00.1 0.2 — — — 75% sf; 3% bevacizumab; 22% sucrose 205-01 480 mb; 1 mm;5% bevst; lyo; 0.0 2.7 2.9 — — — 85% sf; 5% bevacizumab; 10% sucrose205-02 480 mb; 1 mm; 5% bevst; lyo; 0.0 0.6 1.0 — — — 73% sf; 5%bevacizumab; 22% sucrose 202-03 480 mb; 1 mm; 30% st; 5% bevst; 0.0 0.4— — — — lyo; 95% sf; 5% bevacizumab 205-04 480 mb; 1 mm; 40% st; 4° C.;lyo; 0.0 0.3 0.9 0.8 — — 85% sf; 5% igg; 10% sucrose 205-05 480 mb; 1mm; lyo; 95% sf; 0.0 3.3 1.2 2.6 0.6 0.7 5% lysozyme 205-06 480 mb; 1mm; lyo; 85% sf; 0.0 1.0 1.6 1.1 *0.0 1.2 5% lysozyme; 10% sucrose205-07*** 480 mb; 1 mm; lyo; 75% sf; 0.0 2.9 3.0 2.5 2.9 4.5 25%lysozyme 205-08 480 mb; 1 mm; lyo; 65% sf; 0.0 1.1 2.6 2.8 6.1 1.8 25%lysozyme; 10% sucrose 205-A 120 mb; 1 mm; lyo; 95% sf; 0.0 1.1 1.1 0.30.1 — 5% lysozyme 197-09 480 mb; 1 mm; 40% st; lyo; 96.5% sf; 0.0 0.00.0 0.0 0.0 — 2.5% bsa; 1% trehalose 197-11 120 mb; 1 mm; 30% st; lyo;96.5% sf; 0.0 1.8 3.0 8.6 5.6 — 2.5% bsa; 1% trehalose 197-12*** 120 mb;1 mm; 30% st; lyo; 94% sf; 0.0 0.5 0.2 2.2 1.1 — 5% bsa; 1% trehalose201-04 480 mb; 1 mm; 5% bevst; 4° C.; lyo; 0.0 2.5 3.4 2.3 3.0 2.5 95%sf; 5% bevacizumab

Silk fibroin molecular weight seemed to play a role in release oflysozyme from silk fibroin rods. Increasing the silk fibroin molecularweight from low molecular silk fibroin (480 mb) to relatively highermolecular weight silk fibroin (120 mb), with 5% lysozyme loading as seenin samples 205-05 and 205-A respectively, decreased the initial burstand cumulative release percentage over 3 days.

BSA-containing rods with lower molecular weight silk fibroin (480 mb)showed a protein-loading dependent release. Rods prepared from 480 mbsilk fibroin with 2.5% BSA showed release below detectable levels (BDL)out to 3 days (197-09). Rods prepared from 120 mb silk fibroin with lowloading (2.5% BSA, sample 197-11) showed faster release kinetics incomparison with the corresponding rods with higher BSA loading (197-12).The lower loaded 120 mb rods (197-11) initial burst at 2 hours of 13.7%and a cumulative release of 26.1% by day 3, 120 mb silk fibroin rodsshowed faster release of BSA than the comparable formulation made with480 mb silk fibroin (which showed no release). The results suggested arelationship between the BSA:silk fibroin ratio and the release kineticsof the protein from the rod.

For the silk fibroin rods prepared with bevacizumab, all formulationsshowed very little burst (less than or equal to 7%) with no continuedrelease, with the exception of the rod formulation prepared at 4° C.(201-04). This low temperature rod had a burst at 2 hours of 56.0% ofthe loaded protein, with 84.5% of the protein released after 1 day Thisformulation temperature-dependent release could be caused by an increasein non-specific or hydrophobic binding of silk fibroin and bevacizumabat elevated temperatures. The lower temperature might also effect thetightness and size of the silk fibroin network within the rodformulation.

The silk fibroin rod with IgG subject to the in vitro experiments(205-04, 480 mb; 1 mm; 40% st; 4° C.: lyo; 85% sf; 5% igg; 10?% sucrose)showed a lower burst and release out to 2 days. 2 hours into theexperiment, 5.7% of the protein was released, and the cumulative releasepercentage leveled after 1 day at about 19.4%. This rod released moreprotein than similar rods with 5% bevacizumab (205-01), but it releasedless protein than similar rods with 5% lysozyme (205-06).

The release data from 5% analyte rod formulations for lysozyme (205-05),BSA (197-12), and bevacizumab (202-03 and 205-01) demonstrated a trend.The smaller proteins, lysozyme and BSA, had higher burst releases fromthe rods and faster release kinetics than bevacizumab. Additionally, therods formulated with smaller proteins seemed to release protein overseveral days, whereas release of bevacizumab (a larger molecule) for therod formulation plateaued after 1 day of release.

Example 17. Excipient Effects on Release Kinetics of Protein Cargo

Silk fibroin rods were formulated with proteins, and the controlledrelease of said proteins were monitored in vitro. Silk fibroin rods wereformulated with 5 or 25% (w/w) lysozyme (molecular weight=14 kDa). Somesilk fibroin rods were formulated with 5 or 25% (w/w) lysozyme, and with10% (w/w) sucrose as an excipient. The excipient was added to reduce thesilk concentration, while increasing the size of the silk fibroinnetwork and tuning the release kinetics.

Silk fibroin rods were prepared from silk fibroin degummed with a 480mb. Sodium chloride was purchased from Chemsavers (Bluefield Va.).Polysorbate-80 was purchased from Croda (Snaith, United Kingdom).Phosphate buffered saline (10×PBS) was purchased from Gibco (USA).Sodium phosphate dibasic, sodium phosphate monobasic, human lysozyme,sucrose, were purchased from Sigma-Aldrich (St. Louis, Mo.). Sodiumazide and glycerol were purchased from Fisher Chemical (Waltham, Mass.).

Preparation of Silk Fibroin Rods with Proteins and Other Excipients

To prepare the silk fibroin rods with lysozyme, silk fibroin wasdissolved in lysozyme stock solution to reach the final desiredsilk/lysozyme concentrations. Sucrose (Sigma Aldrich, St. Louis Mo.) wasdissolved in this solution when necessary. Formulations were injectedinto 1.0 mm diameter PTFE tubing. The tubing was capped with Parafilm®and allowed to gel at 37° C. overnight. Once gelling was achieved, thetubing was frozen and lyophilized. The formulations were prepared asdescribed in Table 35. The silk fibroin rods are named by the processused to prepare and formulate each rod. For example, the rod named 480mb; 1 mm; lyo; 85% sf; 5% lysozyme; 10% sucrose refers to a rod preparedwith silk degummed with a 480-minute boil (480 mb), a 1 mm diameter(mm), lyophilization (lyo), a theoretical w/w percentage of 85% silkfibroin (85% sf), a theoretical w/w percentage of 5% lysozyme (5%lysozyme), and a theoretical w/w percentage of 10% sucrose (10%sucrose). Sample 205-05 also contained 133.3 mM phosphate buffer.

TABLE 35 Silk rods formulated with proteins and excipients Time of Silk-Sample heating fibroin Protein Excipient No. Sample name (mb) Conc. %Protein conc. % Excipient conc. % 205-05 480 mb; 1 mm; lyo; 480 95Lysozyme 5 — — 95% sf; 5% lysozyme 205-06 480 mb; 1 mm; lyo; 480 85Lysozyme 5 Sucrose 10 85% sf; 5% lysozyme; 10% sucrose 205-07 480 mb; 1mm; lyo; 480 75 Lysozyme 25 — — 75% sf; 25% lysozyme 205-08 480 mb; 1mm; lyo; 480 65 Lysozyme 25 Sucrose 10 65% sf; 25% lysozyme; 10% sucroseIn Vitro Release Profile of Silk Fibroin Rods Formulated with ProteinAPIs and Other Excipients

Silk fibroin rods were cut into 1 cm sections and two sections wereplaced, in triplicate, into 4 mL glass vials. 1 mL of release media wasadded to each vial. Samples were incubated with gentle shaking at 37° C.At 2 hours, 1, 2, 3, 7, 10, 14, 21, and 28 days, 100 μL of release mediawas removed and replaced with 100 μL of fresh release media. Totalprotein released was quantified via size-exclusion chromatography (SEC)using a Waters X-Bridge Protein BEH SEC, 200 Å, 3.5 μm column. Anisocratic flow of mobile phase (100 mM sodium phosphate (Sigma Aldrich,St. Louis Mo.), 200 mM NaCl (Chemsavers. Bluefield Va.) pH 6.8) was runat 0.80 mL/min to elute protein. Protein elution was monitored at 280and 214 nm using an Agilent 1290 HPLC system with a PDA detector.Cumulative percentage of protein released was calculated usingtheoretical loading of the silk fibroin rods.

The cumulative release percentage of each protein was monitored overtime, as seen in Table 36A and Table 36B. The incorporation of sucrosein the silk fibroin rods resulted in a faster release of lysozyme forsome of the rod formulations. The initial burst of lysozyme release wasat least two-fold greater for the rods formulated with sucrose and 25%lysozyme. Furthermore, the cumulative release percentage of lysozyme wasat least about two-fold greater over time when the rods were formulatedwith sucrose and 25% lysozyme. The cumulative release percentage ofprotein released from each rod were measured in triplicate, except forthe specific measurements marked with “*”, which were measured insinglicate. Sample 205-07, marked with “***”, was tested in duplicate.

TABLE 36A In vitro release of Lysozyme from silk-fibroin rods with andwithout an excipient; cumulative percentage (%) of API released SampleDay No. 0 0.083 1 2 3 7 205-05 0.00 18.74 27.19 32.43 7.21 7.64 205-060.00 19.42 29.37 34.84 *29.54 10.29 205-07*** 0.00 17.50 22.42 35.0437.12 39.75 205-08 0.00 48.69 74.98 83.25 71.14 73.94

TABLE 36B Standard deviation of in vitro release of Lysozyme fromsilk-fibroin rods with and without an excipient; in terms of cumulativepercentage (%) of API released Day Sample No. 0 0.083 1 2 3 7 205-050.00 3.25 1.24 2.63 0.56 0.71 205-06 0.00 1.00 1.61 1.09 0.00* 1.24205-07*** 0.00 2.85 2.98 2.47 2.87 4.52 205-08 0.00 1.13 2.60 2.80 6.111.85

Silk fibroin rods loaded with 5% lysozyme (sample 205-05) had similarrelease profiles to rods loaded with 25% lysozyme (205-07). However, theaddition of sucrose affected these formulations very differently.Replacing 10% silk fibroin with sucrose did not change the 5% lysozymeloaded formulation release, while it increased the initial burst(measured at 2 hours) of the 25% lysozyme rod from 17.5% to 48.7%. Thisresult suggested a critical silk fibroin:lysozyme ratio that needed tobe maintained to reduce the initial burst. Adding sucrose in place ofsilk fibroin reduced this ratio enough in the higher loaded lysozymerods, but not in the rods with lower loading.

Example 18. In Vivo Ocular Pharmacokinetic Studies with Silk FibroinRods and Hydrogels with Celecoxib

Silk fibroin platforms were evaluated for delivery of celecoxib (CXB) tothe intraocular tissues. Both the hydrogel and rod formulations werewell tolerated, showing no negative clinical symptoms, rise inintraocular pressure (IOP), or adverse histological findings over 6months. After the silk fibroin rods or 0.050 mL samples of hydrogelswere administered, the SBPs were subject to pharmacokinetic studies.Multiple preparations of the same formulations were used. The averagecalculated CXB dose for the hydrogels comprised 3.5-3.6 mg, while theaverage calculated CXB dose comprised 0.59 to 0.75 mg for the rods.Clinical exams, intraocular pressure (IOP), and histological assessmentwere performed to determine local tolerability. Vitreous humor (VH) andretina/choroid (RC) tissues were collected and analyzed for CXBconcentration over 6 months. Animals had gross examinations of the eyeas well as slit-lamp fundus examinations. For slit-lamp exams, ahand-held slit-lamp (Koma or similar) were used.

Briefly, the concentration of API in the vitreous humor was determinedafter the administration of CXB via silk fibroin rod. After the in vivosilk rods experiments, the vitreous humor of the subjects of theexperiments was analyzed for the concentration of celecoxib present. Thesilk fibroin rods (480 mb; 0.5 mm; 40% st; 100 mgsf; 200 mgcxb; lyo;33.3% sf; 66.7% cxb) and silk fibroin hydrogels were administered to theleft eye of New Zealand white rabbits, with a total celecoxib dose of640-750 sg. Two to three animals were used in each group for each timepoint. The rabbits were sacrificed at about 2 weeks, I month 2 months, 3months, 4.5 months, and 6 months after injection.

Formulation Residence Time

The formulations containing celecoxib were still clinically visible at 6months post injection (10% CXB suspension, 10% CXB hydrogel, and CXBrod). All hydrogel and suspension groups had reduced in size over time.Additionally, the 1.4% CXB suspension was visible clinically out to 3months. A blank hydrogel formulation was evaluated out to 7 months, andalthough it decreased in size, it was still clinically present at thetime of sacrifice. Formulations had no adverse clinical findings for theduration of the study.

Celecoxib Detection in Aqueous Humor

The concentration of API in the aqueous humor was determined after theadministration of CXB with different API delivery media. To collect theaqueous humor, the animals were anesthetized. Approximately 50-100 μLaqueous humor was removed from the anterior chamber at the limbus by a31G needle attached to a 1 mL insulin syringe. Samples of the aqueoushumor were prepared in a 50/50 Acetonitrile/50 mM Ammonium Formate, pH4.0 buffer and analyzed via HPLC. The results of the in vivoadministration of celecoxib through the eye were shown in Table 37. Asseen in the Table 37, at least 50% of the animals subject to experimentswith silk fibroin rods had detectable amounts of CXB in the aqueoushumor after 7 days. 100% of the animals tested with silk fibroin rodshad detectable levels of CXB in the aqueous humor after 28 days.

TABLE 37 Detection and concentration of celecoxib in the aqueous humorafter intraocular administration Average % of Concen- Animals with CXBtration St. Detectable Sample Name Dose Day (ng/mL) Dev. CXB 10% CXBControl 5 mg 7 0.43 0.45 100 28 1.25 1.09 100 56 0.58 0.40 100 480 mb;hyd; 5 mg 7 1.03 1.66 83.3 27.8% cxbst; 28 1.17 0.49 100 3% SFf; 10%CXBf; 56 1.62 0.40 100 10% P188f 480 mb; 0.5 mm; 0.7 mg   7 0.27 0.15 5040% st; 100 mgsf; 28 0.38 0.30 100 200 mgcxb; lyo; 56 0.30 0.07 10033.3% sf; 66.7% cxb

Celecoxib Detection in Whole Eye

The animals were euthanized, and eyes were enucleated and immediatelysnap frozen in liquid nitrogen, position of the implant/formulation wasvisualized and recorded to ensure that each eye was orientedappropriately during freezing and dissection. The eyes were thenbisected ensuring that the implant/formulation was completely retainedin one half of vitreous. The eyes were then thawed, and both vitreoushemispheres (formulation and no formulation) were collected. Thevitreous with no formulation was analyzed for CXB concentration viaHPLC-MS. The vitreous containing the formulation was centrifuged at10,000×g for 10 minutes. The supernatant was removed and analyzed forCXB concentration via HPLC-MS. Samples of the vitreous humor wereprepared in a 50/50 Acetonitrile/50 mM Ammonium Formate, pH 4.0 bufferprior to analysis via HPLC. The formulation pellet collected aftercentrifugation was frozen and lyophilized. CXB was extracted from theformulations using acetonitrile and analyzed via HPLC-UV.

Furthermore, the retina and choroid were dissected from both hemispheresfor extraction and analysis via HPLC-MS. Samples of retinoid wereinitially wetted with acetonitrile and dried prior to samplepreparation. The retinoid samples were finely cut with a scissors andmixed into a uniform paste. 10 times the weight of 50/50 Acetonitrile/50mM ammonium formate pH 4 was added to every sample. The samples werethen vortexed for 2 minutes, sonicated for 15 minutes, and refrigeratedovernight. The samples were then sonicated for an additional 15 minutes,then centrifuged for 8 minutes and then processed per the same testprocedures used for the aqueous and vitreous humors.

The concentration of celecoxib in the vitreous humor from each bisectedhalf (with and without the implanted silk fibroin rod) was analyzed, asseen in Table 38A and Table 38B. At each timepoint, the concentration ofcelecoxib in the vitreous humor, with and without the implant, wasdetermined to be greater than or equal to the IC₅₀, the half-maximalinhibitory concentration, of celecoxib, which was 40 nM (15.3 ng/mL).The silk fibroin rods showed near steady state drug concentrations, withconcentrations in the vitreous humor greater than or equal to the IC₅₀for three months. Controls of celecoxib suspensions were also analyzed,with an approximate dosage of 5 mg celecoxib.

TABLE 38A Descriptions of samples analyzed for concentrations ofcelecoxib in whole eye CXB CXB CXB CXB Theoretical Average TheoreticalAverage Sample Sample Name Dose Dose loading Loading Low 1.4% CXB 0.7 mg(14   0.65 mg 1.4%      1.3% CXB Suspension mg/mL) control High 10% CXB5 mg (100 4.0-4.3 mg 10% 8.0-8.5% CXB Suspension mg/mL) control 10% 480mb; hyd; 5 mg (100 3.5-3.6 mg 10% 6.9-7.2% CXB 27.8% cxbst; mg/mL)hydrogel 3% SFf; 10% CXBf; 10% P188f CXB 480 mb; 0.7 mg 0.59-0.75 mg 66.7%  44.7-52.5%  rods 0.5 mm; 40% st; (N/A) 100 mgsf; 200 mgcxb; lyo;33.3% sf; 66.7% cxb

TABLE 38B Detection and concentration of celecoxib in the vitreous humor(VH) and retina after intraocular administration VH No VH Retina/ SampleCXB Implant Std Implant Std Choroid Std Sample Name Dose Day (ng/mL)Dev. (ng/mL) Dev. (ng/mL) Dev. Low 1.4% CXB 0.7 mg (14 14 817 690 1773329503 4190 4587 CXB Suspension mg/mL) 29 100 129 28806 39590 65 15control 84 17 12 3445 4992 55 48 High 10% CXB 45 mg (100 14 491 787 434665 36338 53177 CXB Suspension mg/mL) 86 11 2 7125 7036 131 37 control127 133 161 1173 462 141 37 170 1998 2760 834 914 1194 154 10% CXB 480mb; hyd; 5 mg (100 14 4663 7314 12167 12262 7349 11480 hydrogel 27.8%cxbst; mg/mL) 86 3807 5124 18050 3182 60400 27153 3% SFf; 127 125 144708 165 344 161 10% CXBf; 170 24 4 1314 1353 122 81 10% P188f CXB rods480 mb; 0.7 mg 14 70 91 413 441 133 99 0.5 mm; (N/A) 29 11832 16501 22013 1254 1635 40% st; 58 25 21 317 80 1493 986 100 mgsf; 86 31 18 783 77460 46 200 mgcxb; 126 30 24 170 97 79 39 lyo; 33.3% sf; 169 45 1 234 4159 11 66.7% cxb

At 14 days, the low concentration suspension formulations exhibitedcomparatively lower CXB concentrations in the vitreous with noformulation, while the vitreous with formulation as well as theretina/choroid had higher concentrations of CXB. This may have been dueto the nature of the suspension formulations, which are more diffusewithin the vitreous humor and more difficult to separate than the silkfibroin formulations. The vitreous humor containing the formulationranged from 28806 ng/mL to 3445 ng/mL CXB, maintaining levels well abovethe estimated EC₈₀ for celecoxib (1-3 μM; 381-1143 ng/mL). The vitreoushumor with no formulation as well as the retina/choroid showed verysimilar trends of high concentration at 14 days followed by a dramaticdrop by 30 days. This low level was decreased further out to 90 days.The intravitreal concentration of CXB generally decreased over the 84day time frame with the administration of the 1.4% CXB suspension. CXBconcentrations in multiple tissues fell below the EC₈₀ by 29 days andapproached the reported biochemical inhibitory concentration (IC50; 40nM; 15 ng/mL) by 90 days post injection.

The intravitreal injection of a 10% CXB suspension showed decreasingretinal tissue concentrations from 14 to 86 days (36338 ng/mL to 131ng/mL). This concentration was then maintained in the retina/choroidover 6 months at 130-200 ng/mL (below the EC₈₀ for celecoxib). Vitreoushumor CXB concentrations displayed differences over time which seemed tobe dependent on the hemisphere. Over the 170 day experiment, theconcentration of CXB delivered by the 10% CXB suspension, was variableamongst the tissues. After injection of the 10% CXB suspension, bothvitreous halves had similar CXB concentrations at 14 days (491 ng/mL and433 ng/mL for no formulation and formulation vitreous respectively);however, these two locations varied more noticeably at the latertimepoints (86 days or longer). The vitreous humor containing theformulation showed a maximum CXB concentration of 7125 ng/mL at 3months, which then decreased to approximately 1000 ng/mL after 127 days.The vitreous humor from the hemisphere containing no formulation droppedto a concentration of only 11 ng/mL at about 3 months, then increased at127 and 170 days to 133 ng/mL and 1998 ng/mL. This variability, similarto the lower concentration suspension group, may have been due to thedispersity of the suspension and inefficient removal of undissolved CXBduring extraction. Although all of the tissues displayed levels at orabove the EC₈₀ for CXB at 14 days, only the vitreous humor containingthe formulation maintained concentrations in this range over the 6months of the study. CXB concentrations in the other tissues fell wellbelow this concentration by 3 months.

The silk-fibroin hydrogel formulation containing 10% CXB (5 mg dose)displayed elevated, steady-state concentrations in both vitreous samplesas well as retina/choroid tissue over 86 days, which decreased slightlythereafter. The retina/choroid showed CXB levels of 7349 ng/mL and 60400ng/mL (7 times and 60 times the EC₈₀ for CXB) at 14 days and 86 days,respectively. Concentrations decreased to 344 ng/mL at 127 days (withinthe EC₈₀) and further to 122 ng/mL at about 6 months. Vitreous humorcontaining the formulation maintained levels at or above the EC₈₀ forthe duration of the study. Over the first 3 months, concentrationsranged slightly from 12167-18050 ng/mL CXB. These concentrationsdecreased to 708 ng/mL and 1314 ng/mL at 127 and 170 days. The vitreoushumor with no formulation was also well above the EC₈₀ over the first 3months with concentrations in the range of 3807-4663 ng/mL. Similar tothe other tissues, CXB concentrations decreased at about 4.5 and 6months, however these CXB levels fell below the EC₈₀. The hydrogelsmaintained higher local levels of CXB over the course of the study.These concentrations were above the IC₅₀ for CXB to COX-2, as describedin Table 39. During the 6 months of the study all tissue concentrationsfor the hydrogel formulation were maintained well above the IC₅₀ forCXB.

Silk-fibroin md implant formulations loaded with CXB exhibitedsteady-state drug levels in the vitreous as well as retina/choroid abovethe IC₅₀ for CXB to COX-2 for greater than 3 months, and at least 169days. Silk-fibroin rod implant formulations loaded with CXB exhibitedsteady-state drug levels in the vitreous humor as well as retina/choroidabove the IC₅₀ for CXB to COX-2 for 6 months. Data showed that the CXBconcentration in the two vitreous humor samples trended together withthe same steady-state. However, in most cases there was 5-10 timeshigher CXB concentration throughout the study in the hemispherecontaining the implant, displaying a CXB concentration gradient.Individual timepoints at 14 days, about 2, about 3, about 4, and about 6months indicated that the CXB concentration in vitreous humor was higherin the hemisphere containing the implant. In the vitreous humorcontaining the implant, CXB levels ranged from 170 ng/mL to 783 ng/mLover the 6 months evaluated, with the highest concentration recorded at86 days. These concentrations were very close to the expected EC₈₀ forCXB. Drug levels in the opposing vitreous humor hemisphere, however,dipped below this mark and ranged from 25 ng/mL to 70 ng/mL, with anexception of 11832 ng/mL at about 1 month. Retina/choroid tissue showeda spike in CXB concentration of 1254 and 1493 ng/mL at 29 and 58 daysrespectively, bringing the levels above the efficacious range (EC₈₀).CXB concentrations in the retina/choroid at 14 days and about 3-6 monthswere lower and very steady, ranging from only 60 ng/mL to 159 ng/mL.

TABLE 39 Fold increase of concentration of celecoxib in the eye overIC₅₀ of celecoxib with COX-2 (In vivo API concentration/IC₅₀ ofcelecoxib) Fold over IC50 (15 ng/mL) (API concentration/IC50 of API withCOX-2) CXB VH No VH Retina/ Sample Sample Name Dose Day Implant ImplantChoroid Low 1.4% CXB 0.7 mg 14 54.5 1182.2 279.3 CXB Suspension 29 6.61920.4 4.4 control 84 1.2 229.6 3.7 High 10% CXB   4 mg 14 32.7 28.92422.5 CXB Suspension 86 0.7 475.0 8.7 control 127 8.9 78.2 9.4 170133.2 55.6 12.9 10% CXB 480 mb; hyd;   5 mg 14 310.9 811.1 489.9hydrogel 27.8% cxbst; 86 253.8 1203.3 4026.7 3% SFf; 127 8.3 47.2 22.910% CXBf; 170 1.6 87.6 8.1 10% P188f CXB rods 480 mb; 0.7 mg 14 4.6 27.58.9 0.5 mm; 40% st; 29 788.8 14.7 83.6 100 mgsf; 58 1.7 21.1 99.5 200mgcxb; lyo; 86 2.0 52.2 4.0 33.3% sf; 126 2.0 11.3 5.2 66.7% cxb 169 3.015.6 10.6

The administration of the silk fibroin compositions resulted in in vivoconcentrations of CXB consistently above the IC₅₀ of celecoxib with itstarget protein, COX-2 (40 nM or 15 ng/mL). The administration of eitherthe silk fibroin hydrogels or the rods resulted in a higher intraocularconcentration of CXB near the ocular area of administration (e.g. thehalf of the eye in which the rod was positioned). The intraocularconcentrations of CXB remained greater than the IC₅₀ of CXB over thecourse of the experiment. The silk fibroin hydrogels sustainedintraocular concentrations of CXB greater than the estimated EC80 (1-3μM or 381-1143 ng/mL) for the first 86 days. About 3 months afterhydrogel administration, the intraocular CXB concentration lowers, butit remains above the IC₅₀ for CXB for the remainder of the study. Thesilk rods delivered a lower, more consistent concentration of CXB overtime in comparison with the hydrogels.

Regardless of proximity of the formulation to the area of the eye or theamount of time since injection, the silk fibroin hydrogel or rodcompositions resulted in CXB concentrations at least 1.7-fold greaterthan the IC₅₀ in the vitreous humor and at least 4-fold greater than theIC₅₀ in the retina/choroid over the first 86 days. Over the course of169 or 170 days, the silk fibroin rod or hydrogel compositions resultedin CXB concentrations at least 1.6-fold greater than the IC₅₀ in thevitreous humor and at least 4-fold greater than the IC₅₀ in theretina/choroid.

Over the first 86 days, administration of the hydrogels resulted in aconcentration at least 250-fold greater than the IC₅₀ of celecoxib inthe vitreous humor without the implant, at least 800-fold greater thanthe IC₅₀ of celecoxib in the vitreous humor with the implant, and atleast 480-fold greater than the IC₅₀ of celecoxib in the retina/choroid.Over 170 days, administration of the hydrogels resulted in aconcentration at least 1.6-fold greater than the IC₅₀ of celecoxib inthe vitreous humor without the implant, at least 47-fold greater thanthe IC₅₀ of celecoxib in the vitreous humor with the implant, and atleast 8-fold greater than the IC₅₀ of celecoxib in the retina/choroidover the course of the experiment.

Over the first 86 days, administration of the rods resulted in aconcentration at least 1.7-fold greater than the IC₅₀ of celecoxib inthe vitreous humor without the implant, at least 14-fold greater thanthe IC₅₀ of celecoxib in the vitreous humor with the implant, and atleast 4-fold greater than the IC₅₀ of celecoxib in the retina/choroid.Over 169 days, administration of the rods resulted in a concentration atleast 1.7-fold greater than the IC₅₀ of celecoxib in the vitreous humorwithout the implant, at least 11-fold greater than the IC₅₀ of celecoxibin the vitreous humor with the implant, and at least 4-fold greater thanthe IC₅₀ of celecoxib in the retina/choroid.

Both the hydrogel and the rod were able to deliver CXB at or above theEC₈₀, concentration of compound needed to elicit 80% of a completeresponse. The EC₈₀ was estimated to be 1-3 μM for CXB in this system.Hydrogel administration resulted in intraocular concentrations of CXBabove the EC₈₀ for the first 86 days, but the intraocular concentrationof CXB was at or below the efficacious range after 86 days. Rodadministration resulted in intraocular concentrations at or near theefficacious range in the vitreous humor with the formulation for thefirst 86 days. The hydrogel platform was able to deliver CXB atconcentrations at least 3 times the EC₈₀ for less than or equal to 3months in all the ocular tissues.

Both the rod and hydrogel formulations showed residence in theintraocular space for at least 6 months. The results indicated thatsilk-fibroin hydrogels and silk-fibroin rod implants were bothwell-tolerated formulation options that maintained steady-state deliveryof CXB to ocular tissues for at least 3-6 months. Even with the majordifferences in CXB dose (5 mg in the hydrogel; 700 μg in the rod), CXBlevels were maintained in the back of the eye above the IC₅₀ for CXB toCOX-2 over the course of the study. This indicated that theconcentrations were in an efficacious range.

Example 19. Macromolecular Therapeutic Agent Storage and Stability by aSilk Composition Silk Fibroin Isolation and Hydrogel Formation

Silk yam is degummed at 100° C. for 120 minutes in 0.02 M sodiumcarbonate aqueous solution to remove sericin. 30 g of cut silk yam isboiled in 1 L of deionized (DI) water with 0.02 M sodium carbonate for80 minutes under stirring. Then the yam is transferred to a new boiling0.02 M sodium carbonate aqueous solution and boiled for additional 40minutes under stirring. The fibroin is then placed in DI water at 60-70°C. for 20 minutes under stirring, and then rinsed with clean DI water.This is repeated three times. The fibroin is then placed in clean DIwater and stirred for 20 minutes, then rinsed with clean DI water andrepeated for a total of three 20 minute-rinse cycles. The fibroin isthen dried overnight, weighed, and dissolved at 20% (w/v) in a 9.3 Maqueous solution of lithium bromide for 5 hours at 60° C. The resultingfibroin solution is dialyzed against water at 4° C. in a 50 kDaregenerated cellulose dialysis tubing for 48 hours with 6 water changesto remove the excess salt. The conductivity is recorded after each waterchange with a digital quality tester. When the conductivity is under 5ppm the fibroin is ready.

The solution is centrifuged three times for 20 minutes each at 9,000 RPMand 4° C. to remove insoluble particles. The supernatant is collected,and samples of the supernatant are diluted at 1:20 and 1:40 in water.Standard samples are prepared for an A280 assay by diluting pre-measuresfibroin solutions to 5.2.5, 1.25, 0.625, 0.3125, and 0 mg/mL in water,for the generation of a standard curve. The silk concentration of the1:20 and 1:40 diluted silk fibroin samples is measured against thestandard curve using absorbance at 280 nm.

The fibroin solutions are diluted to a final concentration of 3% (w/v)in 10 mM phosphate buffer or TRIS buffer, pH 7.4. Some solutions of silkfibroin are also prepared with 0.5-5% (w/v) sucrose and/or 2-10 mMhistidine buffer. The solutions are filtered through a 0.2 μm filterusing a vacuum filter unit. Sucrose can be added to the solution priorto freezing to aid in reconstitution of the lyophilized silk fibroinafter lyophilization. Then, 10 mL of each solution is aliquoted into 50mL conical tubes, snap frozen in liquid nitrogen for 10 minutes,transferred for 20 minutes in −80° C., and lyophilized for 72 hours.

Therapeutic Agent Loading in Silk Fibroin Hydrogel

Lyophilized silk fibroin is dissolved with a solution of the therapeuticagent to obtain concentrations of 1.3, 3.6, 7.0, 13.0, and 23.0% (w/v)silk fibroin. A gelling agent (PEG400, glycerol, Poloxamer, etc.) isadded to the therapeutic/silk solution to induce gel formation. The tubecan be left at 4° C., room temperature (RT) or 37° C. overnight toinduce gelation.

Stability of Therapeutic Agent

The effect of silk fibroin hydrogel on the stability of the therapeuticagent is evaluated by placing samples of the therapeutic loaded silkfibroin hydrogel at different temperatures (4° C., 25° C. or 37° C.). Atweekly timepoints, the therapeutic agent is extracted from theformulation by placing a known mass of the formulation into a compatiblebuffer. The extracted solution is analyzed by using a stabilityindicating HPLC assay as well as a cell-based activity assay. Thestructural integrity of the formulation and/or the therapeutic agent isdetermined by using an HPLC assay and evaluating the presence ofaggregation. The functional activity of the therapeutic is evaluated byusing a cell-based assay.

In Vitro Release

An aliquot of the fibroin-therapeutic hydrogel is added to a 2-mLEppendorf tube. 1.95 mL of release medium (PBS, pH 7.4) is added. Thesamples are incubated at 37° C. with gentle shaking. The release mediumis changed after 24 hours and then approximately once daily for 7 days.The release medium is analyzed by HPLC to determine therapeuticconcentration. A calibration curve is generated for the therapeuticagent by dissolving a known amount of the therapeutic agent in therelease medium.

Example 20. Macromolecular Therapeutic Agent Storage and Stability bySilk Fibroin Solutions

Lyophilized silk fibroin is dissolved in water to obtain concentrationsof 1.3, 3.6, 7.0, 13.0, and 23.0% (w/v) silk fibroin. These silk fibroinsolutions are used as stock solutions to prepare therapeutic solutionscomprising 0.1%-30% silk fibroin and a therapeutic agent. Thetherapeutic solution is formulated with excipients and buffers includingthe silk fibroin solution.

The effect of the silk fibroin solutions on the stability of thetherapeutic agent is evaluated by placing solutions of the therapeuticsolutions containing silk fibroin at different temperatures (4° C., 25°C. or 37° C.). At weekly timepoints, the therapeutic solution isanalyzed by using a stability indicating HPLC assay as well as acell-based activity assay. The HPLC assay determines structuralintegrity of the formulation by evaluating the presence of aggregation.The functional activity of the therapeutic agent is evaluated by using acell-based assay.

Example 21. Macromolecular Therapeutic Agent Lyophilization Stability bySilk Fibroin

Lyophilized silk fibroin is dissolved in water to obtain concentrationsof 1.3, 3.6, 7.0, 13.0, and 23.0% (w/v) silk fibroin. These silk fibroinsolutions are used as stock solutions to prepare therapeutic solutionscomprising 0.1%-30% silk fibroin and a therapeutic agent. Thetherapeutic agent is formulated with excipients and buffers includingthe silk fibroin solution. These solutions are then placed in glassvials, frozen and lyophilized.

The effect of silk fibroin solutions on the stability of the therapeuticagent through lyophilization is evaluated by placing the lyophilizedvials of the therapeutic containing silk fibroin at differenttemperatures (4° C., 25° C. or 37° C.). At weekly timepoints, thetherapeutic formulation is reconstituted. The reconstituted solution isanalyzed by using a stability indicating HPLC assay as well as acell-based activity assay. The HPLC assay determines the structuralintegrity of the formulation by evaluating the presence of aggregation.The functional activity of the therapeutic agent is evaluated by using acell-based assay.

Example 22. Release Characteristics of Celecoxib from Silk FibroinHydrogels of Varying Silk Fibroin Molecular Weights

Silk yam was purchased from Jiangsu SOHO Silk and Textile Co. (Jiangsu,China). Lithium Bromide was purchased from Sigma-Aldrich (St. Louis,Mo.). Polysorbate-80 was purchased from Croda (Snaith, United Kingdom).The potassium phosphate monobasic and the potassium phosphate dibasicwere purchased from Sigma Aldrich Fine Chemicals (St. Louis, Mo.). Theglycerol, sodium carbonate, and sodium azide were purchased from FisherChemical (Waltham, Mass.). The celecoxib (CXB) was purchased from Cipla(Miami, Fla.).

Silk Fibroin Isolation

Silk yarn from SOHO was degummed at 100° C. for either 30, 60, 90,120,or 480 minutes in 0.02 M sodium carbonate solution to remove sericin andmodify fibroin molecular weight. The amount of boiling time was referredto as the “minute boil” or “mb”. Longer boiling times produced silkfibroin with smaller molecular weights. 480 mb silk fibroin has anaverage molecular weight of between 30-60 kDa, 120 mb silk fibroin hasan average molecular weight of between 100-300 kDa, and 90 mb silkfibroin has an average molecular weight of about 361 kDa. Fibroin wasdried overnight, weighed, and dissolved at 20% (w/v) in 9.3 M lithiumbromide solution for five hours at 60° C. The resulting solution wasdialyzed against water in a 50 kDa regenerated cellulose membrane for 48hours at 4° C. with six water changes. The resulting solution wascentrifuged for 20 minutes at 9,000 RPM and 4° C. to remove insolubleparticles. Solutions were diluted to a final concentration of 3% (w/v)in 10 mM phosphate buffer, pH 7.4, filtered through a 0.22 μm filter,frozen in liquid nitrogen, and lyophilized for at least 72 hours.Lyophilized silk fibroin was stored at −20° C. or less prior to use.

Hydrogel Preparation

Lyophilized silk-fibroin was reconstituted to a concentration of 6%(w/v) using a suspension of celecoxib. The silk/CXB suspension had afinal concentration of 6% (w/v) silk-fibroin, 20% (w/v) CXB insuspension, 0.2% polysorbate-80, and 44 mM phosphate buffer. Silk/CXBand 80% glycerol in water solutions were then combined at a ratio of 1:1and mixed until homogeneous. The final formulation for all hydrogelsprepared was: 3% (w/v) silk-fibroin, 40% glycerol, 10% CXB, 0.1%tween-80, and 22 mM phosphate buffer, pH 7.4. Gels were incubated at 37°C. on an orbital mixer overnight to induce gelation, and the hydrogelswere stored at 4° C. until use. The formulations tested were named bythe method in which they were prepared. For example, in the sample named480 mb; hyd; 3% SFf; 10% CXBf; 40% Glyc, “480 mb” refers to silkdegummed with a 480-minute boil, “hyd” refers to the formulation of thesample as a hydrogel, “3% SFf” refers to a formulation with 3% (w/v)silk fibroin, “10% CXBf” refers to a formulation with 10% (w/v)celecoxib, and “40% Glyc” refers to a formulation with 40% (w/v)glycerol. Some samples were prepared with silk fibroin degummed with a120, 90, 60, or 30-minute boil (120 mb, 90 mb, 60 mb, and 30 mbrespectively). The formulations were listed in Table 40. In Table 40,“PS-80” is Polysorbate-80.

TABLE 40 Formulations of silk fibroin hydrogels prepared from silkfibroin degummed with different boiling times for the cumulative releaseexperiments Actual Silk CXB conc. ± boiling Silk TPS-80 PhosphateGlycerol CXB Standard Sample time conc. conc. Buffer conc. conc.Deviation Sample name (mb) (% w/v) (% w/v) (mM) (% w/v) (% w/v) (% w/v)No. 480 mb; hyd; 480 3 0.1 22 40 10 10.92 ± 161-1 3% SFf; 0.31 10% CXBf;40% Glycf 120 mb; hyd; 120 3 0.1 22 40 10 9.78 ± 161-2 3% SFf; 0.22 10%CXBf; 40% Glycf 90 mb; hyd; 90 3 0.1 22 40 10 9.27 ± 161-3 3% SFf; 1.7210% CXBf; 40% Glycf 60 mb; hyd; 60 3 0.1 22 40 10 9.19 ± 161-4 3% SFf;0.52 10% CXBf; 40% Glycf 30 mb; hyd; 30 3 0.1 22 40 10 9.34 ± 161-5 3%SFf; 0.78 10% CXBf; 40% Glycf Solution N/A 0 0.1 22 40 10 11.68 ± 161-6control 0.67

In Vitro Release of Celecoxib

In triplicate, 50 mg of each formulation was weighed into half of a #4gelatin capsule. Capsules were placed into a 50 mL, conical tubecontaining 45 mL of release medium (Ix phosphate buffered saline. 2%Polysorbate-80, and 0.05% sodium azide). The solubility of celecoxib inthis release media is 850 μg/mL. 45 mL of this release media allows for38 mg CXB solubility. This media ensured sink conditions (greater thanor equal to 5 times CXB solubility) were maintained throughout thecourse of the study. The tubes were capped and incubated at 37° C. withshaking. 1 mL of the release media was collected from each sample atdays 1,4, 7,10, 14 and 21 days and replaced with fresh media. At eachtimepoint, the tubes were stood on end for at least 30 minutes, to allowthe formulation to settle prior to taking the sample. Release media wasanalyzed by HPLC-UV (Agilent 1290 Infinity) at 260 nm. Controls wereprepared at Day 0 by weighing 50 mg of each formulation in triplicate inseparate 20 mL. glass vials. Methanol was added to each sample toextract CXB. Samples were placed on a shaker at room temperature for 24hrs. The supernatant was analyzed by HPLC-UV to determine CXB loading.The results of the release studies were displayed in Table 41A and Table41B.

TABLE 41A In vitro release kinetics for silk fibroin hydrogels withvarying molecular weight silk loaded with celecoxib; average cumulativepercentage (%) of API released Sample Day No. 0 1 3 7 14 21 28 161-1 0.069.6 93.0 89.1 91.6 — — 161-2 0.0 26.9 49.0 66.8 82.2 91.9 86.4 161-30.0 28.9 54.6 74.2 90.1 100.3  94.1 161-4 0.0 27.5 51.3 67.0 81.6 89.083.8 161-5 0.0 47.7 71.7 78.1 86.3 91.7 86.4 161-6 0.0 70.9 95.1 93.793.0 — —

TABLE 41B Standard Deviation of in vitro release kinetics for silkfibroin hydrogels with varying molecular weight silk loaded withcelecoxib; in terms of average cumulative percentage (%) of API releasedSample Day No. 0 1 3 7 14 21 28 161-1 0.0 7.2 1.8 3.3 7.0 — — 161-2 0.02.2 1.2 2.0 1.3 2.8 2.6 161-3 0.0 2.0 0.5 3.3 4.5 5.7 5.5 161-4 0.0 1.82.6 3.9 5.1 6.1 5.8 161-5 0.0 21.9 12.1 6.0 4.6 6.5 6.2 161-6 0.0 5.64.6 4.3 5.5 — —

The 480 mb hydrogels approached 100% CXB the quickest following asimilar trajectory to the CXB suspension alone. This was most likely dueto the formulation not completely gelling. When placed in release mediait did not hold its shape and it dispersed as a suspension. Formulationsprepared with the higher molecular weight range of silk-fibroindisplayed similar release profiles following first-order releasekinetics, with an initial burst of approximately 30% out to 21 days,with the exception of the hydrogel made with highest silk-fibroinmolecular weight (30 mb). This formulation displayed a slightly higherburst than the others, but the release continued out to 21 days.

Example 23. Rheological Characteristics of Celecoxib-containingSilk-Fibroin Hydrogels of Varying Silk Fibroin Molecular Weights

Silk yam was purchased from Jiangsu SOHO Silk and Textile Co. (Jiangsu,China). Lithium Bromide was purchased from Sigma-Aldrich (St. Louis,Mo.). Polysorbate-80 was purchased from Croda (Snaith, United Kingdom).The potassium phosphate monobasic and the potassium phosphate dibasicwere purchased from Sigma Aldrich Fine Chemicals (St. Louis, Mo.). Theglycerol, sodium carbonate, and sodium azide were purchased from FisherChemical (Waltham, Mass.). The celecoxib (CXB) was purchased from Cipla(Miami, Fla.). Silk fibroin isolation

Silk yam from SOHO was degummed at 100° C. for either 30, 60, 90, 120,or 480 minutes in 0.02 M sodium carbonate solution to remove sericin andmodify fibroin molecular weight. The amount of boiling time was referredto as the “minute boil” or “mb”. Longer boiling times produced silkfibroin with smaller molecular weights. 480 mb silk fibroin has anaverage molecular weight of between 30-60 kDa, 120 mb silk fibroin hasan average molecular weight of between 100-300 kDa, and 90 mb silkfibroin has an average molecular weight of about 361 kDa. Fibroin wasdried overnight, weighed, and dissolved at 20% (w/v) in 9.3 M lithiumbromide solution for five hours at 60° C. The resulting solution wasdialyzed against water in a 50 kDa regenerated cellulose membrane for 48hours at 4° C. with six water changes. The resulting solution wascentrifuged for 20 minutes at 9,000 RPM and 4° C. to remove insolubleparticles. Solutions were diluted to a final concentration of 3% (w/v)in 10 mM phosphate buffer, pH 7.4, filtered through a 0.22 μm filter,frozen in liquid nitrogen, and lyophilized for at least 72 hours.Lyophilized silk fibroin was stored at −20° C. or less prior to use.

Hydrogel Preparation

Lyophilized silk-fibroin was reconstituted to a concentration of 6%(w/v) using a suspension of celecoxib. The silk/CXB suspension had afinal concentration of 6% (w/v) silk-fibroin, 20% (w/v) CXB insuspension, 0.2% polysorbate-80, and 44 mM phosphate buffer. Silk/CXBand 80% glycerol in water solutions were then combined at a ratio of 1:1and mixed until homogeneous. The final formulation for all hydrogelsprepared was: 3% (w/v) silk-fibroin, 40% glycerol, 10% CXB, 0.1%polysorbate-80, and 22 mM phosphate buffer, pH 7.4. Gels were incubatedat 37° C. on an orbital mixer overnight to induce gelation, and thehydrogels were stored at 4° C. until use. The formulations tested werenamed by the method in which they were prepared. For example, in thesample named 480 mb; hyd; 3% SFf; 10% CXBf 40% Glyc, “480 mb” refers tosilk degummed with a 480-minute boil, “hyd” refers to the formulation ofthe sample as a hydrogel, “3% SFf” refers to a formulation with 3% (w/v)silk fibroin, “10% CXBf” refers to a formulation with 10% (w/v)celecoxib, and “40% Glyc” refers to a formulation with 40% (w/v)glycerol. Some samples were prepared with silk fibroin degummed with a120, 90, 60, or 30-minute boil (120 mb, 90 mb, 60 mb, and 30 mbrespectively). The formulations were listed in Table 42.

TABLE 42 Formulations of silk fibroin hydrogels prepared from silkfibroin degummed with different boiling times for the rheologicalexperiments Silk boiling Silk Polysorbate- Phosphate Glycerol CXB timeconc. 80 conc. Buffer conc. conc. Sample name (mb) (% w/v) (% w/v) (mM)(% w/v) (% w/v) 480 mb; hyd; 3% SFf; 480 3 0.1 22 40 10 10% CXBf; 40%Glyc 120 mb; hyd; 3% SFf; 120 3 0.1 22 40 10 10% CXBf; 40% Glyc 90 mb;hyd; 3% SFf; 90 3 0.1 22 40 10 10% CXBf; 40% Glyc 60 mb; hyd; 3% SFf; 603 0.1 22 40 10 10% CXBf; 40% Glyc 30 mb; hyd; 3% SFf; 30 3 0.1 22 40 1010% CXBf; 40% Glyc

Rheological Measurements of Silk Fibroin Formulations

The hydrogel samples were loaded onto a Peltier plate system held at 25°C. The geometry used was a 20 mm parallel plate with a gap of 1 mm andfrequency at 1 Hz. Viscosity was measured during a time sweep at 1 s-1over 135 seconds. The storage modulus (G′), the loss modulus (G″), andthe phase angle were then measured during a time sweep over 145 secondsat 0.1% strain and 1 Hz. As seen in Table 43, the rheology showed ageneral increase in viscosity from silk fibroin prepared from a longerboiling time (480 mb) to silk fibroin prepared from a shorter boilingtime (30 mb); therefore, the viscosity increased from low molecularweight silk-fibroin to high molecular weight silk-fibroin formulations.In Table 43. “Std. Dev.” refers to standard deviation.

TABLE 43 Rheological properties of silk fibroin hydrogels withcelecoxib. Viscosity Phase G′ G″ Boil Std. Phase Angle Std. Std. SampleTime Viscosity Dev. Angle Std. Dev. G′ Dev. G″ Dev. No. (mb) (Pa*s)(Pa*s) (°) (°) (Pa) (Pa) (Pa) (Pa) P00161- 480 6.56 1.67 9.60 2.97 76.988.43 12.91 3.85 01 P00161- 120 49.72 2.81 8.43 0.43 1148.30 93.06 169.698.63 02 P00161- 90 65.25 2.25 8.88 0.48 1652.94 134.85 257.58 13.09 03P00161- 60 118.64 6.55 12.41 0.68 4279.45 276.60 939.41 45.63 04 P00161-30 169.61 7.40 14.78 1.22 7820.86 539.69 2057.36 145.74 05

The viscosities ranged from 7 to 170 Pa s-1 for the range of molecularweights tested. The stiffness (as measured by G′ and G″, seen in Table43) also showed an increase with increasing molecular weight ofsilk-fibroin, as defined by the minute boil. The phase angle, as seen inTable 43, increased slightly for the hydrogel formulations prepared fromsilk fibroin with a shorter boiling time. As the molecular weight of thesilk-fibroin increased (marked by a lower degumming time) the hydrogelformulations were stiffer and much more viscous. These results displayedthe range of properties the silk-fibroin hydrogel formulations couldhave. The formulations had also been used to analyze the release of CXBover time, and the physical characteristics of the hydrogels were ableto be modified while only minimally affecting release kinetics.

Example 24. Rheology Studies of Silk Fibroin Hydrogels

Hydrogel samples were loaded into a Peltier plate system, with a 20 mmparallel plate geometry, at a temperature of 25° C. The gap was set to 1mm, and the frequency was set to 1 Hz. Viscosity measurements weremeasured with a shear ramp was from 0.1 1/s to 1 1/s over 113 s with 11samples, followed by a shear hold at 1 1/s for 180 s with 18 samples.Oscillatory measurements were measured with a strain ramp from 0.01 to1% strain with a constant 1 Hz frequency over 173 s with 21 measurementsand the G′, G″, and phase angle were averaged over the linearviscoelastic region (LVR). The viscosity was first studied as a functionof silk fibroin concentration, as seen in Table 44. The viscosity of thesilk fibroin hydrogels was studied for hydrogels with two differentexcipients. Silk fibroin hydrogels were studied with silk fibroinconcentrations of 6%, 5%, 4%, 3%, and 2% (w/v) silk fibroin degummedwith a 120-minute boil. The hydrogels were prepared with either 40%PEG300 or 40% glycerol. 0.2% polysorbate-80, 22 mM phosphate buffer, and10% celecoxib (CXB). The components of the gel were mixed and allowed togel at 37° C. with rotation.

TABLE 44 Rheological properties of silk fibroin hydrogels with varyingconcentrations of silk fibroin Standard Deviation Average of the AverageSilk Phase Phase Sample Fibroin Excip. G′ G″ Angle Visc. G′ G″ AngleVisc. No. % Excip. % (Pa) (Pa) (°) (Pa*s) (Pa) (Pa) (°) (Pa*s) 130-01 6PEG300 40 71188 12346 10.13 1798 30242 4469 0.53 687 130-02 6 Glycerol40 80647 12307 8.77 1722 46411 6745 0.39 957 130-03 5 PEG300 40 332975859 10.04 717 8723 1426 0.27 184 130-04 5 Glycerol 40 33737 5054 8.54726 12631 1873 0.16 275 130-05 4 PEG300 40 21504 3845 10.24 364 81241409 0.48 142 130-06 4 Glycerol 40 18618 2677 8.21 379 6331 886 0.11 111130-07 3 PEG300 40 4968 996 11.52 57 440 101 2.12 1 130-08 3 Glycerol 407511 1046 7.95 161 2977 410 0.15 68 130-09 2 PEG300 40 2484 473 11.05 341923 365 1.29 26 130-10 2 Glycerol 40 1814 257 8.24 31 1915 264 0.27 18

The viscosity of the hydrogels increased with the concentration of silkfrom.

Example 25. Formulation and Release Characteristic of Rods of IncreasedHydrophilicity

SBPs were formulated as rods to determine whether soluble and/or bulkyadditives to silk fibroin rod formulations would increase API release.These additives were also included to enhance and increase the rate ofin vivo degradation of silk fibroin rods. The silk fibroin was degummedfor 480 minutes. The formulations tested were named by the method inwhich they were prepared. For example, in the sample named “480 mb; 0.5mm, 20% st; 50 mgsf; 200 mgcxb; oven; 14.8% sf; 59.3% cxb; 25.9%sucrose/poly-20” refers to a silk fibroin rod prepared from silkdegummed with a 480-minute boil (480 mb), an extrusion with a 0.5 mmdiameter (0.5 mm), a preparation from a 20% stock solution of silkfibroin (20% st), a preparation from 50 mg of silk fibroin (50 mgsf), apreparation from 200 mg of celecoxib (200 mgcxb), oven drying (oven), atheoretical w/v percentage of 14.8% silk fibroin (14.8% sf), atheoretical w/v percentage of 59.3% celecoxib (59.3% cxb), and atheoretical w/v percentage of 25.9% other additives such as sucrose andpolysorbate-20 (25.9% sucrose/poly-20). The samples tested were listedin Table 45. Other additives tested included polysorbate-80 (poly-80),trehalose, mannitol, PEG 2 kDa, hydroxyethylcellulose (HEC),carboxymethylcellulose (CMC), polyvinylpyrrolidone K-17 (K17), andpolyvinylalcohol (PVA). The term theoretical loading percentage refersto the assumed percentage of a component incorporated in a substance orproduct. The product may be an SBP.

TABLE 45 Formulations of silk fibroin hydrogels prepared with variousfillers to alter hydrophilicity Theoretical Theoretical TheoreticalSample Formulation dry CXB dry SF dry other number Description Name (mg)(mg) (mg) 222-01 40% SF; 480 mb; 0.5 mm; 40% st; 100 mgsf; 200 100 0Control; Oven 200 mgcxb; oven; 33.3% sf; Dried 66.7% cxb 222-03 20% SF;70% 480 mb; 0.5 mm; 20% st; 50 mgsf; 200 50 87.5 Sucrose + 0.5% 200mgcxb; oven; 14.8% sf; Polysorbate-20; 59.3% cxb; 25.9% sucrose/poly-20Oven Dried 222-05 20% SF; 70% 480 mb; 0.5 mm; 20% st; 50 mgsf; 200 5087.5 Sucrose + 0.5% 200 mgcxb; oven; 14.8% sf; Polysorbate-80; 59.3%cxb; 25.9% sucrose/poly-80 Oven Dried 222-09 20% SF; 70% 480 mb; 0.5 mm;20% st; 50 mgsf; 200 50 87.5 Trehalose; Oven 200 mgcxb; oven; 14.8% sf;Dried 59.3% cxb; 25.9% trehalose 222-11 20% SF; 70% 480 mb; 0.5 mm; 20%st; 50 mgsf; 200 50 87.5 Trehalose + 200 mgcxb; oven; 14.8% sf; 0.5%59.3% cxb; 25.9% trehalose/poly-80 Polysorbate-80; Oven; Dried 222-1520% SF; 70% 480 mb; 0.5 mm; 20% st; 50 mgsf; 200 50 87.5 Mannitol + 200mgcxb; oven; 14.8% sf; 0.5% 59.3% cxb; 25.9% mannitol/poly-80Polysorbate-80; Oven Dried 222-17 20% SF; 50% 480 mb; 0.5 mm; 20% st; 50mgsf; 200 50 62.5 PEG 2 kDa; 200 mgcxb; oven; 50% 16.0% sf; Oven Dried64.0% cxb; 20.0% PEG2 kDa 222-19 20% SF; 5% 480 mb; 0.5 mm; 20% st; 50mgsf; 200 50 6.25 HEC + 0.05% 200 mgcxb; oven; 19.5% sf; Polysorbate-20;78.0% cxb; 2.4% hec/poly-20 Oven Dried 222-21 20% SF; 5% 480 mb; 0.5 mm;20% st; 50 mgsf; 200 50 6.25 CMC + 0.05% 200 mgcxb; oven; 19.5% sf;Polysorbate-20; 78.0% cxb; 2.4% cmc/poly-20 Oven Dried 222-25 20% SF;20% 480 mb; 0.5 mm; 20% st; 50 mgsf; 200 50 25 K17 + 0.05% 200 mgcxb;oven; 18.2% sf; Polysorbate-20; 72.7% cxb; 9.1% k17/poly-20 Oven Dried222-27 20% SF; 5% 480 mb; 0.5 mm; 20% st; 50 mgsf; 200 50 6.25 PVA +0.05% 200 mgcxb; oven; 19.5% sf; Polysorbate-20; 78.0% cxb; 2.4%pva/poly-20 Oven Dried

The density of the experimental loadings as well as the densities of thesilk fibroin rods were also determined, as seen in Table 45. Thedifferences in theoretical and experimental loadings of celecoxib werealso determined as a percentage of the theoretical w/w loading ofcelecoxib. In Table 46, “Std. Dev.” refers to standard deviation.

TABLE 46 Experimental loadings and densities of silk fibroin rods withincreased hydrophilicity % Difference between Std. Dev. theoretical Std.Sample Experimental % Experimental % of exp. % and actual Density Dev.of number SF CXB CXB loading of CXB (g/mL) Density 222-01 36.44 63.562.83 −5%  1.09 0.03 222-03 38.07 61.93 1.16 5% 1.03 0.04 222-05 36.5863.42 3.60 7% 0.96 0.07 222-09 45.41 54.59 3.43 −8%  1.06 0.04 222-1136.95 63.05 1.19 6% 1.11 0.06 222-15 26.57 73.43 1.64 24%  0.92 0.01222-17 39.10 60.90 2.44 −5%  1.13 0.08 222-19 19.87 80.13 3.69 3% 0.820.04 222-21 20.24 79.76 5.44 2% 0.85 0.06 222-25 19.51 80.49 3.84 11% 0.87 0.07 222-27 20.26 79.74 3.88 2% 0.86 0.01

The silk fibroin rods were subject to in vitro release experiments todetermine the release kinetics of celecoxib from these formulations. Thesilk fibroin rods were incubated in PBS with 0.6% polysorbate-80 and0.05% sodium azide over the course of the experiment. The averagecumulative release percentage of celecoxib overtime was depicted in therelease kinetics shown in Table 47A and Table 47B.

TABLE 47A In vitro release kinetics for hydrophilic silk fibroin rodsloaded with celecoxib; average cumulative percentage (%) of API releasedSample Number 222- 222- 222- 222- 222- 222- 222- 222- 222- 222- 222- CXBDay 01 03 05 09 11 15 17 19 21 25 27 suspension 0 0.0 0.0 0.0 0.0 0.00.0 0.0 0.0 0.0 0.0 0.0 0.0 1 12.7 18.7 17.1 21.4 18.1 16.1 20.1 21.216.4 18.6 20.5 106.8 4 31.9 44.4 47.7 50.7 44.6 45.6 48.3 55.4 44.4 53.049.5 110.1 7 46.3 60.8 63.0 64.4 60.3 61.8 67.3 71.4 57.7 67.7 60.4 91.614 55.0 70.2 74.5 77.0 72.7 75.3 80.1 82.9 70.7 82.6 74.8 87.7 21 63.378.6 92.0 86.7 82.5 85.2 92.2 81.7 85.2 85.0 84.7 86.5 28 81.5 93.5 91.794.5 99.2 99.0 100.9 96.3 97.6 98.8 93.7 90.4 35 83.5 91.4 88.8 91.294.7 94.4 97.1 88.5 92.1 89.1 89.3 83.3 42 88.6 90.3 88.7 91.4 92.5 95.097.2 89.0 92.0 92.4 87.6 — 49 92.1 91.3 89.2 92.5 95.0 95.1 99.3 88.992.7 87.8 87.7 — 56 93.6 92.2 89.8 93.9 96.4 96.6 99.1 90.6 93.5 94.789.7 —

TABLE 47B Standard deviation of average cumulative percentage of APIreleased in vitro for hydrophilic silk fibroin rods loaded withcelecoxib Sample Number 222- 222- 222- 222- 222- 222- 222- 222- 222-222- 222- CXB Day 01 03 05 09 11 15 17 19 21 25 27 suspension 0 0.0 0.00.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1 0.9 0.7 0.4 2.7 1.6 0.7 1.61.8 1.0 0.5 3.6 1.0 4 2.6 1.3 3.0 3.6 4.3 1.9 4.0 2.8 2.6 1.9 2.4 1.4 75.5 3.2 1.3 3.5 7.1 0.8 4.8 4.8 4.8 4.6 4.0 6.5 14 3.0 2.3 1.5 1.4 8.12.2 3.3 5.7 3.4 3.5 1.3 3.7 21 2.9 2.5 18.4 6.1 10.7 0.8 2.1 6.7 9.1 1.55.2 14.6 28 3.5 1.2 2.6 0.2 10.4 5.7 5.8 3.2 3.5 3.1 2.8 15.5 35 2.4 2.41.8 0.8 9.7 4.5 5.6 7.5 3.7 6.1 0.2 16.9 42 3.8 1.8 2.4 0.0 14.2 4.3 6.57.4 3.5 2.3 2.8 — 49 3.9 2.3 1.6 0.5 9.5 4.7 6.3 7.8 2.5 8.7 3.3 — 562.9 2.3 1.7 0.2 10.8 4.8 6.2 7.8 3.1 2.3 2.6 —

Overall, formulations with additives, including sucrose, trehalose,mannitol, polysorbate-20, polysorbate-80, PEG 2 kDa, HEC, K17, CME, andPVA, showed increased API release during the initial burst as comparedto silk fibroin rods without the additives. As used herein, the term“initial burst” refers to a rate of factor release from a source ordepot over an initial release period (e.g., after administration orother placement, for example in solution during experimental analysis)that is higher than rates during one or more subsequent release periods.The initial burst was evaluated at 1 day for the silk fibroin rods. Thesilk fibroin rods with additives also demonstrated increased API releaseover the first 35 days of the experiment as compared to silk fibroinrods without the additives. These data suggested that additives to silkfibroin rods can be used to tune API release kinetics. The additivesmight also assist in rod degradation in vivo. The faster the drug isreleased from the formulation, the faster the majority of the surfacearea is exposed to the environment, and theoretically the faster thesilk fibroin will degrade from enzymatic degradation. The control rodtakes more time to disperse all of the API, and therefore will be aroundlonger than rods that disperse API in less time.

Example 26. Analysis of Celecoxib Remaining in Silk Fibroin Rods afterIn Vivo Administration

After the in vivo silk rods experiments, the silk fibroin rods wereanalyzed for the amount of celecoxib (CXB) that remained. At the desiredtimepoints of the in vivo experiments, New Zealand white rabbits weresacrificed, and their eyes were enucleated, snap frozen, and bisected.The formulation, hydrogel or implant (480 mb; 0.5 mm; 40% st; 100 mgsf;200 mgcxb: lyo; 33.3% sf; 66.7% cxb) was removed from the eyes andcollected for further studies. The vitreous containing the formulationwas centrifuged at 10,000×g for 10 minutes. The resulting formulationpellet was frozen and lyophilized. Any remaining celecoxib was extractedfrom the formulations using acetonitrile and analyzed via HPLC-UV.Briefly, the formulation pellets were brought up in acetonitrile, andthen vortexed, sonicated, and left on a shaker at room temperature for24 to 48 hours. The supernatant was filtered through a 0.2 μm nylonsyringe filter, diluted and then analyzed via HPLC-UV. The percentage ofcelecoxib remaining in the rod was studied as a function of time of invivo study, as seen in Table 48.

TABLE 48 Celecoxib remaining in CXB loaded silk rods after intravitrealinjection Average % St. Dev. Sample Sample Name Day Remaining (%) LowCXB 1.4% CXB 14 65.0 19.9 control Suspension 29 31.3 31.6 86 53.9 68.6High CXB 10% CXB 14 0.4 0.6 control Suspension 86 88.8 3.6 127 139.239.6 170 87.0 0.6 10% CXB 480 mb; hyd; 14 100.3 20.2 hydrogel 27.8%cxbst; 86 49.9 17.2 3% SFf; 10% CXBf; 127 87.0 22.5 10% P188f 170 81.67.9 CXB rods 480 mb; 0.5 mm; 14 72.5 2.0 40% st; 100 mgsf; 29 45.3 7.1200 mgcxb; lyo; 58 75.1 3.0 33.3% sf; 86 53.6 22.1 66.7% cxb 126 52.15.9 169 10.0 4.0

The concentration of celecoxib remaining in the rods decreased linearlyas time passed. Extractions performed on the silk fibroin rods over thecourse of the study displayed a zero-order release of celecoxib in thevitreous. Fitting a curve to this linear regression demonstrated a goodfit with the exception of the 1 month timepoint. The data demonstratedthat approximately 10% of the loaded CXB still remained in the implantat 6 months. The in vivo half-life of release of the CXB from the rodimplant, which represented the amount of time required for 50% of thecelecoxib to be released from the silk fibroin rod, was estimated to be3.5 months (about 85 days), with 90% CXB released by 6 months (169days). Recoveries of the API from the hydrogel showed that there wasstill significant API remaining after completion of the study. Theextractions of CXB from the rods and the hydrogels demonstrated thatthere was still sufficient CXB remaining to maintain steady-statedelivery for at least 6 months with a single administration, since morethan 50% of the celecoxib remained after 3 months of the experiment.Furthermore, the silk fibroin rods released CXB at a rate faster thanthat of the silk fibroin hydrogels in vivo.

Example 27. Histopathology Studies of Rabbit Eyes with Silk RodsCompared with Silk Hydrogels

Eight formalin-fixed rabbit eyes were submitted to HistoTox Labs andprocessed into two blocks per sample. Eyes with gel formulations werecollected at 203 days, and eyes with rod formulations were collected at117 days. One slide per block was sectioned and stained with hematoxylinand eosin (H&E). Glass slides were evaluated by a board-certifiedveterinary pathologist using light microscopy. The presence of injectedmaterial was recorded, and histologic lesions were graded for severity(0=absent: 1=minimal; 2=mild; 3=moderate; 4=marked; 5=severe). Theresults of the experiment were summarized in Table 49. In Table 49, “P”refers to present and “NP” refers to not present.

TABLE 49 H&E grades of the rabbit eye histopathology data of animalstreated with silk fibroin rod and hydrogel compositions H&E InjectedInfiltration, Retinal material, mononuclear cell/ distortion/ vitreousmultinucleated cell, degeneration, Treatment Sample Block chamberinjected material focal Silk-Fibroin CCN- 1 NP — 0 Hydrogel 43L 2 P 0 0(acellular aggregate) CCN- 1 NP — 0 44L 2 NP — 0 CCN- 1 NP — 0 45L 2 P 00 (acellular aggregate) Silk-Fibroin/ CCN- 1 NP — 0 CXB Rod 86L 2 P(rod) 1 2 CCN- 1 P (rod) 0 0 87L 2 P (rod) 0 2 CCN- 1 NP — 0 88L 2 NP —0 Untreated CCN- 1 NP — 0 43R 2 NP — 0 CCN- 1 NP — 0 86R 2 NP — 0

Injected material was visible in most injected (left) eyes. Injectedsilk fibroin hydrogel material was visible in two of three injectedeyes; this material formed a mass up to 5 mm in diameter in the vitreouschamber, composed of pale amphophilic granular material surrounding50-200 μm diameter pale basophilic structures with a more solidappearance. This material consistently lacked cellular infiltrates whencaptured. There were no other histologic findings in the silk fibroinhydrogel-injected eyes.

Injected material consistent with silk fibroin/celecoxib (CXB) rod wasvisible in two of the three injected eyes. This structure was present inthe vitreous chamber, in close proximity to the retina; it wasapproximately 500 μm diameter, stained basophilic to amphophilic, andcontained non-staining vacuoles or clefts. In one sample (CCN-86L. Block1), the rod structure was surrounded and infiltrated by lymphocytes,macrophages, and multinucleated giant cells; however, in all otherinstances the rod was acellular. In two samples, the retina adjacent tothe rod was focally distorted, with disorganized retinal layers and cellvacuolization. Given the proximity to the injected rod, this lesion wasconsidered to be secondary to the injection procedure.

Example 28. Physical Properties of Silk Fibroin Hydrogels with Celecoxibfor In Vivo Studies

Silk fibroin hydrogels were prepared as described above. Briefly,lyophilized silk fibroin was reconstituted with an aqueous solution ofsodium chloride, polysorbate-80, and phosphate buffer. The sodiumchloride concentration was adjusted to ensure a final osmolarity of 280mOsm. A suspension of celecoxib (CXB) was used to reconstitute silkfibroin in these hydrogel formulations. The silk fibroin was allowed tofully reconstitute prior to being drawn into a 6 mL syringe. Excipientsolutions were prepared so that a 0.75:1 mix of silk-fibroinsolution:excipient solution would result in the desired finalformulations. The pH of polyethylene glycol (PEG) hydrogels was adjustedusing hydrochloric acid to account for the changes in pH observed whenmixing phosphate buffer and PEG. The excipient solutions were drawn upinto a second 6 mL syringe. The solutions were mixed back and forth viaa syringe connector until homogeneous. The resulting mixture wasincubated at 37° C. overnight and aliquoted into 1 mL syringes prior toinjection.

The formulations were prepared as described in Table 50. Multiplepreparations of the same formulation may be examined. The samples inTable 50 were named by the process used to prepare and formulate eachhydrogel. For example, the sample named “I 20 mb; hyd; 27.8% cxbst; 3%SFf; 10% CXBf; 10% P188f” refers to a formulation prepared from silkfibroin degummed with a 120-minute boil (120 mb), in a hydrogel format(hyd), from a stock of 27.8% w/v celecoxib (27.8% cxbst), with 3% w/vsilk fibroin (3% SFf), with 10% w/v celecoxib (10% CXBf), and with 10%P188 (10% P188f). Longer boiling times (mb) produced silk fibroin withsmaller molecular weights.

TABLE 50 Properties of hydrogel formulations with celecoxib Sample No.169-2 169-3 Sample name 120 mb; hyd; 27.8% 480 mb; hyd; 27.8% cxbst; 3%SFf; 10% cxbst; 3% SFf; 10% CXBf; 10% P188f CXBf; 10% P188f Description10% CXB; 3% 120 mb 10% CXB; 3% 480 mb Silk; 10% Silk; 10% Poloxamer-188Poloxamer-188 Average Actual 9.61 9.77 CXB % Average CXB 4.8 4.9 dose(mg) pH 7.06 7.15 Viscosity (Pa*s) 76.44 113.16 Phase Angle (°) 5.358.68 G′ (Pa) 4487.2 9117.6 G″ (Pa) 418.9 1384.7 Injection force 8.1 9.9(N) at 0.2 mL/minute

Rheological Analysis of Hydrogel Formulations

The theological properties of the hydrogel samples were analyzed. Usinga Bholin CVOR 150 rheometer, 800 μL of each sample was directlydeposited onto a Peltier Plate system using a 25 mm diameter parallelplate. The oscillation method kept strain, temperature, and frequencyconstant at 0.1%, 25° C., and 1 Hz respectively. A time sweep was usedto measure the G′, G″, and phase angle values over 150 seconds. Theviscoelastic method kept the shear rate, strain, and frequency constantat I 1/s, 0.1%, and 1 Hz respectively. A time sweep then measured theviscosity over 60 seconds. This was performed in triplicate for eachsample.

The results of the experiments were shown in Table 50. The hydrogel withlower molecular weight silk fibroin (480 mb) had a higher viscosity andphase angle than the hydrogel with higher molecular weight silk fibroin(120 mb). Indeed, the viscosity of sample 169-3 (480 mb; hyd; 27.8%cxbst; 3% SFf; 10% CXBf; 10% P188f) was measured at 113.16 Pa*s,approximately 1.5 times greater than the measured viscosity of sample169-2 (120 mb; hyd; 27.8% cxbst; 3% SFf; 10% CXBf; 10% P188f) at 76.44Pa*s. The phase angle of sample 169-3 (480 mb; hyd; 27.8% cxbst; 3% SFf;10% CXBf; 10% P188f) was 8.68°, approximately 1.6 times the phase angleof sample 169-2 (120 mb; hyd; 27.8% cxbst; 3% SFf, 10% CXBf; 10% P188f)at 5.35°.

The hydrogel with lower molecular weight silk fibroin (480 mb) also hada higher shear storage modulus and shear loss modulus than the hydrogelwith higher molecular weight silk fibroin (120 mb). As used herein, theterm “shear storage modulus” or “G′” refers to the measure of amaterial's elasticity or reversible deformation as determined by thematerial's stored energy. As used herein, the term “shear loss modulus”or “G″” refers to the measure of a material's ability to dissipateenergy, usually in the form of heat. Sample 169-3 (480 mb; hyd; 27.8%cxbst; 3% SFf; 10% CXBf; 10% P188f) had a G′ value and a G″ value of9117.6 Pa and 1384.7 Pa respectively. Sample 169-2 (120 mb; hyd; 27.8%cxbst; 3% SFf; 10% CXBf; 10% P188f) had a G′ value and a G″ value of4487.2 Pa and 418.9 Pa respectively. The measured G′ for the lowermolecular weight hydrogel was twofold greater than that of the highermolecular weight silk fibroin hydrogel, while the measured G″ was atleast threefold greater than that of the higher molecular weight silkfibroin hydrogel. Ultimately, the use of lower molecular weight silkfibroin produced thicker, more viscous gels.

Injection Forces

The force required to extrude the hydrogels was measured. Each hydrogelsample was mixed back and forth between two syringes to ensurehomogeneity before being loaded into 1 mL syringe and capped with 27G,½″ needles. The syringes were inserted into a Mark-10 syringecompression fixture and the test stand was set to move the head downonto the syringe plunger and extrude the hydrogel at a rate of 0.5in/min This was estimated to be equivalent to 0.2 mL/min with thissyringe configuration. The force gauge measured the force required toextrude the hydrogel with a maximum force set at 200 N. Data wascollected over 60 seconds (20 points per second) and exported andgraphed to find where the injectability force plateau. The average valuewas taken over this plateau region. Each sample was injected intriplicate and average force measurements were calculated. The averageforce measurements were listed in Table 50. The average force forextrusion was measured to be 9.9 N for sample 169-3 (480 mb; hyd; 27.8%cxbst; 3% SFf; 10% CXBf; 10% P188f) and 8.1 N for sample 169-2 (120 mb;hyd; 27.8% cxbst; 3% SFf; 10% CXBf; 10% P188f). Preparation from thelower molecular weight silk fibroin resulted in a stiffer hydrogel thatrequired a greater force for extrusion.

Example 29. Release of Protein Cargo from Silk Fibroin Hydrogels

Silk fibroin hydrogels were prepared from silk fibroin degummed with a480 mb or a 120 mb. Sodium chloride was purchased from Chemsavers(Bluefield Va.). Polysorbate-80 was purchased from Croda (Snaith, UnitedKingdom). Phosphate buffered saline (10×PBS) was purchased from Gibco(USA). Sodium phosphate dibasic, sodium phosphate monobasic, humanlysozyme, sucrose, Bovine Serum Albumin (BSA), trehalose, andpoloxamer-188 (P188) were purchased from Sigma-Aldrich (St. Louis, Mo.).Sodium azide and glycerol were purchased from Fisher Chemical (Waltham,Mass.). Bevacizumab was purchased from Genentech Inc. (San Francisco,Calif.). Human immunoglobulin G (IgG) was purchased from InnovativeResearch (Novi, Mich.).

Silk Fibroin Hydrogel Preparation with Protein

To prepare the hydrogels with lysozyme, purified silk fibroin with a480-minute boil or silk fibroin with a 120-minute boil werereconstituted to a concentration of 30% (w/v) with either water orlysozyme stock solution. The gelation excipient was mixed with thesesolutions to final formulation concentrations. The formulation was drawninto a syringe, capped, and left to gel at 4° C. overnight. Solutionsthat did not gel overnight were transferred to 37° C. for 3 hours toachieve gelling.

To prepare hydrogels with bovine serum albumin (BSA), 300 mg of purifiedsilk fibroin with a 480-minute boil (mb) and silk fibroin degummed witha 120 mb was reconstituted with 0.7 mL of deionized water to make afinal 30% (w/v) solution. BSA was dissolved with either polysorbate-80(PS80) or poloxamer-188 (P188). Solutions were mixed to reach thedesired final concentrations of fibroin/BSA/excipient. The resultingmixture was drawn into a 1 mL syringe, capped, and left to gel at 4° C.overnight. Solutions that did not gel overnight were transferred to 37°C. for 3 hours to achieve gelation.

To prepare hydrogels with bevacizumab, purified silk fibroin degummedwith a 480-minute boil was reconstituted with sufficient deionized waterto a concentration of 10% or 30% (w/v). Bevacizumab was lyophilizedseparately and re-dissolved in the silk solution. An 80% glycerolsolution was mixed with the protein solution to obtain the finalformulations. The resulting mixture was then drawn into a syringe,capped, and left to gel at 4° C. overnight. Solutions that did not gelwere transferred to 37° C. for 3 hours to achieve gelling.

Purified 480 mb silk fibroin and 120 mb silk fibroin were reconstitutedto 30% (w/v) with deionized water. IgG was dissolved with aqueoussolutions of either polysorbate-80 (PS80) or P188. Solutions were mixedto reach the desired final concentrations of fibroin/IgG/excipient. Theresulting mixture was drawn into a syringe, capped, and left to gel at4° C. overnight. Solutions that did not gel overnight were transferredto 37° C. for 3 hours to achieve gelling.

The hydrogels prepared are described in Table 51. The samples were namedfor the process in which they were prepared. For example the ample named“120 mb; hyd; 15% SFf; 2.5% bsaf; 10% PI88f” refers to a sample preparedfrom silk fibroin degummed with a 120-minute boil (120 mb), formulationas hydrogel (h(d), formulation with 15% w/v silk fibroin (15% SFf) aformulation with 2.5 w/v BSA (2.5 bsaf), and formulation with 10% w/P188(10% P188f). Other potential components described included a formulationwith lysozyme (% lysozymef), a preparation from silk fibroin degummedwith a 480 mb (480 mb) a formulation with glycerol (% Glycf), aformulation with bevacizumab (% bevacizumabf), and a formulation withIgG (% iggf).Sample 203-03 (120 mb; hyd; 5% SFf; 2.5% lysozyme; 40%Glycf) did not form a gel. In Table 51, “Excip.” refers to excipient.All IgG hydrogels contained 0.01% polysorbate-80. All lysozyme hydrogelscontained 0.01% polysorbate-80. Bevacizumab hydrogels contained traceamounts of that buffer in which it is provided (trehalose, a sodiumphosphate buffer, and polysorbate-20). All BSA hydrogels contained 0.1%polysorbate-80.

TABLE 51 Preparations of silk fibroin hydrogels and controls withprotein. Silk [Silk Sample [Protein] Fibroin fibroin] [Excip.] Mass No.Protein (%) mb (%) Excip. (%) Sample name (mg) 203-01 Lysozyme 2.5 120 5P188 10 120 mb; hyd; 145.37 5% SFf; 182.93 2.5% lysozymef; 202.12 10%P188f 203-02 Lysozyme 10 120 5 P188 10 120 mb; hyd; 141.1  5% SFf;194.03 10% lysozymef 163.08 10% P188f 203-03 Lysozyme 2.5 120 5 Glycerol40 120 mb; hyd; — 5% SFf; — 2.5% lysozymef; — 40% Glycf 203-04 Lysozyme10 120 5 Glycerol 40 120 mb; hyd; 213.35 5% SFf; 218.72 10% lysozymef;217.83 40% Glycf 203-05 Lysozyme 2.5 120 15 P188 10 120 mb; hyd; 173.3515% SFf; 207.02 2.5% lysozymef; 199.35 10% P188f 203-06 Lysozyme 10 12015 P188 10 120 mb; hyd; 144.88 15% SFf; 207.63 10% lysozymef; 206.52 10%P188f 203-07 Lysozyme 2.5 120 15 Glycerol 40 120 mb; hyd; 223.87 15%SFf; 205.3  2.5% lysozymef; 218.84 40% Glycf 203-08 Lysozyme 10 120 15Glycerol 40 120 mb; hyd; 152.39 15% SFf; 207.88 10% lysozymef; 207.8740% Glycf 203-09 Lysozyme 2.5 480 5 P188 10 480 mb; hyd; 248.13 5% SFf;191.39 2.5% lysozymef; 207.16 10% P188f 203-10 Lysozyme 2.5 480 5Glycerol 40 480 mb; hyd; 209.86 5% SFf; 231.13 2.5% lysozymef; 231.0140% Glycf 203-11 Lysozyme 2.5 480 15 P188 10 480 mb; hyd; 222.07 15%SFf; 210.8  2.5% lysozymef; 234.87 10% P188f 203-12 Lysozyme 2.5 480 15Glycerol 40 480 mb; hyd; 280.46 15% SFf; 223.15 2.5% lysozymef; 232.3740% Glycf 197-01 BSA 2.5 480 5 P188 10 480 mb; hyd; 194.95 5% SFf;194.89 2.5% bsaf; — 10% P188f 197-02 BSA 2.5 480 15 Pl 88 10 480 mb;hyd; 231.42 15% SFf; 226.19 2.5% bsaf; 277.73 10% P188f 197-03 BSA 2.5120 5 P188 10 120 mb; hyd; 203.5  5% SFf; 234.64 2.5% bsaf; 227.49 10%P188f 197-04 BSA 2.5 120 15 P188 10 120 mb; hyd; 252.32 15% SFf; 200.162.5% bsaf; 217.13 10% P188f 197-05 BSA 2.5 120 5 Glycerol 40 120 mb;hyd; 202.99 5% SFf; 225.44 2.5% bsaf; 195.8  40% Glycf 197-06 BSA 2.5120 15 Glycerol 40 120 mb; hyd; 338.31 15% SFf; 206.25 2.5% bsaf; 214.3 40% Glycf 187-2A BSA 2.5 480 5 Glycerol 40 480 mb; hyd; 200.06 5% SFf;206.71 2.5% bsaf; 196.31 40% Glycf 187-4A BSA 2.5 480 15 Glycerol 40 480mb; hyd; 204.86 15% SFf; 207.03 2.5% bsaf; 196.56 40% Glycf 201-01Bevacizumab 2.5 480 5 Glycerol 40 480 mb; hyd; 204.8  5% SFf; 216.912.5% bevacizumabf; 224.1  40% Glycf 201-02 Bevacizumab 2.5 480 15Glycerol 40 480 mb; hyd; 222.22 15% SFf; 225.4  2.5% bevacizumabf;228.86 40% Glycf 201-03 Bevacizumab 2.5 120 5 Glycerol 40 120 mb; hyd;209.93 5% SFf; 190.21 2.5% bevacizumabf; 226.91 40% Glycf 193-01 IgG 2.5480 5 P188 10 480 mb; hyd; 204.54 5% SFf; 197.27 2.5% iggf; 196.44 10%P188f 193-02 IgG 2.5 120 5 P188 10 120 mb; hyd; 192.16 5% SFf; 191.092.5% iggf; 223.79 10% P188f 193-03 IgG 2.5 480 5 Glycerol 40 480 mb;hyd; 201.41 5% SFf; 220.71 2.5% iggf; 205.86 40% Glycf 193-04 IgG 2.5120 5 Glycerol 40 120 mb; hyd; 194.62 5% SFf; 195.54 2.5% iggf; 221.2240% Glycf 193-05 IgG 2.5 480 15 P188 10 480 mb; hyd; 192.2  15% SFf;208.87 2.5% iggf; 226.44 10% P188f 193-06 IgG 2.5 120 15 P188 10 120 mb;hyd; 211.77 15% SFf; 211.43 2.5% iggf; 242.67 10% P188f 193-07 IgG 2.5480 15 Glycerol 40 480 mb; hyd; 228.47 15% SFf; 211.99 2.5% iggf; 241.5740% Glycf 193-08 IgG 2.5 120 15 Glycerol 40 120 mb; hyd; 190.38 15% SFf;215.59 2.5% iggf; 200.78 40% Glycf 1, 2-C Control N/A 120 5 P188 10 120mb; hyd; 242.33 sample 5% SFf; 183.7  10% P188f — 3, 4-C Control N/A 1205 Glycerol 40 120 mb; hyd; 205.27 sample 5% SFf; 204.63 40% Glycf — 5,6-C Control N/A 120 15 P188 10 120 mb; hyd; — sample 15% SFf; — 10%P188f — 7, 8-C Control N/A 120 15 Glycerol 40 120 mb; hyd; 177.8  sample15% SFf; 229.1  40% Glycf — 199-9 BSA 0.025 N/A N/A Glycerol 4 0.025%bsaf; — 4% Glycf — — 199-10 BSA 0.025 N/A N/A P188 1 0.025% bsaf; — 1%P188f — —In Vitro Release Profile of Silk Fibroin Hydrogels Formulated withProtein APIs and Other Excipients

Protein loaded silk-fibroin hydrogels were weighed in triplicate (atapproximately 200 mg) into 4 mL vials. 2 mL of release media were added(PBS, 0.01% polysorbate-80, 0.05% sodium azide). Samples were incubatedwith gentle shaking at 37° C. At 2 hours, 4 hours, 1, 2, 3, 7, 9, 10,14, 21, and 28 days, 150 μL of release media was removed and replacedwith 150 μL of fresh media. Control samples containing 2.5% lysozyme,2.5% IgG, 2.5% bevacizumab, or 2.5% BSA with either 4% glycerol or 1%P188 were prepared to serve as a 100% drug release control. Controlswith protein and gelling agent were utilized to assess the effects ofthe gelling agent on protein stability. Total protein released wasquantified via size-exclusion chromatography using a Waters X-BridgeProtein BEH SEC, 200 Å, 3.5 μm column. An isocratic flow of mobile phase(100 mM sodium phosphate, 200 mM NaCl, pH 6.8) was run at 0.80 mL/min toelute protein. The HPLC system used was an Agilent 1290 with a PDAdetector. Protein elution was monitored at 280 and 214 nm using a PDAdetector. Cumulative % released was calculated using theoreticalloading. Control sample 5,6-C was not tested because it was too stiff toget out of the syringe. The results of the cumulative release studiescould be seen in Table 52A and Table 52B. The samples or readingsdenoted with “*” were completed in duplicate and samples or readingdenoted with “**” were completed in singlicate.

TABLE 52A In vitro release of proteins from silk fibroin hydrogels;average cumulative release percentage (%) of API released each day ofmeasurement Sample Day Protein No. 0 0.08 0.166 1 2 3 5 7 9 Lysozyme203-1 0.0 —  84.575 71.01 95.24 — 96.25 121.36 108.19 Lysozyme 203-2 0.0— 61.3 77.7 111.8 — 117.7 110.4  119.3 Lysozyme 203-4 0.0 — 60.0 65.697.6 — 95.5 88.6 95.8 Lysozyme 203-5 0.0 — 43.4 38.4 58.5 — 69.4 60.465.1 Lysozyme 203-6 0.0 — 76.4 79.8 110.8 — 110.9 107.9  120.8 Lysozyme203-7 0.0 — 22.5 23.1 42.9 — 42.9 44.6 48.4 Lysozyme 203-8 0.0 — 32.473.1 80.4 — 85.7 82.9 89.7 Lysozyme 203-9 0.0 47.8 — 69.6 68.7 69.4 —68.3 — Lysozyme 203-10 0.0 83.1 — 74.0 81.8 82.7 — 83.0 — Lysozyme203-11 0.0 54.6 — 60.6 59.6  54.5** — 52.9 — Lysozyme 203-12 0.0 25.3 —24.4 20.7 20.4 — 19.5 — BSA 197-1* 0.0 — 66.0 131.9 109.1 — — — — BSA197-2 0.0 — 90.4 123.2 105.3 — — — — BSA 197-3 0.0 — 73.0 122.9* 89.7 —— — — BSA 197-4 0.0 — 96.2 136.9* 108.4 — — — — BSA 197-5 0.0 — 77.6121.6 116.9 — — — — BSA 197-6 0.0 — 94.3 103.2 86.6* — — — — BSA 187-2A0.0 — 52.6 74.6* 75.7* — — — — BSA 187-4A 0.0 — 70.8 69.1 68.0 — — — —BSA 199-9 0.0 — 107.9  118.9 119.7 — — — — BSA 199-10* 0.0 — 107.4 124.8 125.6 — — — — Bevacizumab 201-1 0.0 — 36.8 46.6 48.6 46.7 — 43.4 —Bevacizumab 201-2 0.0 — 39.1 45.7 43.7 36.7 — 31.5 — Bevacizumab 201-30.0 — 46.4 72.5 63.1 57.0 — 66.6 — Bevacizumab 201-5 0.0 — 89.9 108.889.2 83.4 — 86.6 — Bevacizumab 201-6* 0.0 — 90.0 103.8 92.4 88.9 — 92.7— IgG 193-01* 0.0 24.1 — 35.6 30.4 — — — — IgG 193-02 0.0  9.8 — 13.812.7 — — — — IgG 193-03 0.0 56.1 — 70.3 59.1 — — — — IgG 193-04 0.0 50.4* — 62.5 50.7 — — — — IgG 193-05 0.0 40.0 — 46.7 45.3 — — — — IgG193-06 0.0 29.0 — 28.5 27.3 — — — — IgG 193-07 0.0 43.0 — 42.0 38.1 — —— — IgG 193-08 0.0 61.1 — 44.9 43.2 — — — —

TABLE 52B Standard deviations of In vitro release of proteins from silkfibroin hydrogels; standard deviations of average cumulative releasepercentage (%) of API released each day of measurement Sample DayProtein No. 0 0.08 0.166 1 2 3 5 7 9 Lysozyme 203-1 0.0 — 16.6 15.4 20.9— 22.3 9.0 22.6 Lysozyme 203-2 0.0 — 3.9 9.4 16.2 — 10.2 16.1 17.6Lysozyme 203-4 0.0 — 4.3 8.2 6.8 — 8.8 7.1 7.7 Lysozyme 203-5 0.0 — 16.415.1 21.3 — 4.2 19.2 21.0 Lysozyme 203-6 0.0 — 16.7 15.7 23.2 — 29.324.4 30.5 Lysozyme 203-7 0.0 — 2.4 5.0 1.8 — 1.9 2.2 2.3 Lysozyme 203-80.0 — 29.1 21.9 5.7 — 4.8 5.9 6.0 Lysozyme 203-9 0.0 5.0 — 13.2 13.611.2  — 12.7 — Lysozyme 203-10 0.0 0.3 — 9.7 5.6 0.4 — 1.2 — Lysozyme203-11 0.0 3.7 — 2.7 3.8  0.0** — 1.4 — Lysozyme 203-12 0.0 1.2 — 1.22.2 0.6 — 0.8 — BSA 197-1* 0.0 — 1.7 0.4 0.4 — — — — BSA 197-2 0.0 —10.0 25.2 7.6 — — — — BSA 197-3 0.0 — 16.3 13.2* 28.6 — — — — BSA 197-40.0 — 10.3 18.3* 14.2 — — — — BSA 197-5 0.0 — 8.4 2.0 28.4 — — — — BSA197-6 0.0 — 15.3 14.1 28.4* — — — — BSA 187-2A 0.0 — 1.9 1.9* 0.5* — — —— BSA 187-4A 0.0 — 6.7 7.4 7.5 — — — — BSA 199-9 0.0 — 3.5 2.1 1.7 — — —— BSA 199-10* 0.0 — 3.7 3.4 3.6 — — — — Bevacizumab 201-1 0.0 — 0.9 0.30.6 0.8 — 0.9 — Bevacizumab 201-2 0.0 — 0.9 1.0 1.3 2.7 — 1.3 —Bevacizumab 201-3 0.0 — 1.6 1.6 2.9 11.9  — 0.9 — Bevacizumab 201-5 0.0— 2.4 0.5 2.5 3.9 — 3.2 — Bevacizumab 201-6* 0.0 — 0.0 3.2 2.4 1.4 — 1.4— IgG 193-01* 0.0 2.6 — 1.2 1.1 — — — — IgG 193-02 0.0 0.7 — 1.2 1.6 — —— — IgG 193-03 0.0 4.0 — 2.5 1.1 — — — — IgG 193-04 0.0 0.2 — 0.7 3.4 —— — — IgG 193-05 0.0 6.5 — 10.6 11.2 — — — — IgG 193-06 0.0 2.2 — 1.82.1 — — — — IgG 193-07 0.0 0.1 — 0.3 0.7 — — — — IgG 193-08 0.0 1.8 —2.3 2.0 — — — —

Lysozyme loading was used to modulate release kinetics. Formulationswith lysozyme and P188 were analyzed first. Formulations prepared withP188 and 10% lysozyme loading and either 5% or 15% 120 mb silk fibroinday (203-2 and 203-6 respectively) reached nearly 80% release by 1 day.120 mb silk fibroin hydrogel formulations with P188 showed silk fibroinconcentration dependent API release. For example, sample 203-1 with 2.5%lysozyme and 5% 120 mb silk fibroin released 84.6% of the API in 4hours. Increasing the silk fibroin concentration to 15% in sample 203-5decreased the release at 4 hours to 43.4%, and caused the release toplateau at approximately 70% over 9 days.

In the hydrogels formulated with P188, the 5% 480 mb silk fibroinhydrogels with 2.5% lysozyme (203-9) showed lower burst and release whencompared to the corresponding 120 mb silk fibroin hydrogels (203-1). Theformulations with P188 and 480 mb silk fibroin also displayed a silkfibroin concentration dependence in release rate with silk fibroinconcentration. This suggested that the release of lysozyme was relatedto the ratio of silk fibroin to lysozyme. The ratios of silk fibroin tolysozyme ranged from 0.5 to 6. In general, an increased ratio of silkfibroin to lysozyme reduced burst and release of the protein. Also,lower molecular weight silk fibroin may form a tighter hydrogel network,further reducing diffusion of the small lysozyme protein.

The release of lysozyme from silk hydrogels prepared with glyceroldisplayed similar trends to the those of the hydrogels prepared fromP188. High loaded glycerol formulations (with 10% lysozyme) with 120 mbsilk fibroin showed a high initial burst release dependent on silkfibroin concentration; higher concentrations of silk fibroin resulted inlower bursts of protein release. The formulation containing lower silkfibroin concentration (lower silk fibroin to lysozyme ratio) reachedapproximately 100% release at 2 days (sample 203-4), while theformulation containing higher concentration of silk fibroin plateaued at80% and continued to release out to 9 days (sample 203-8). Increasingthe silk fibroin to lysozyme ratio by reducing the lysozymeconcentration from 10% to 2.5% reduced the initial burst (measured at 4hours) from 32.4% in sample 203-8 to 22.5% in sample 203-7. This sameeffect can be seen with the 480 mb silk fibroin hydrogel formulations.Increasing the 480 mb silk fibroin concentration from 5% to 15%, whilekeeping the lysozyme loading constant at 2.5%, decreased the initialburst (measured at 2 hours) from 83.1% in sample 203-10 to 25.3% insample 203-12. Lastly, hydrogels with glycerol and with the same silkfibroin to lysozyme ratio and different mb of silk fibroin showedsimilar release kinetics for the first day, however the 120 mb silkfibroin hydrogel (203-7) released at a faster rate over 9 days comparedto the 480 mb silk fibroin hydrogel (203-12). The ratios of silk fibrointo lysozyme ranged from 0.5 to 6 for these hydrogels.

BSA loaded SF hydrogels showed very high burst and complete release ofthe protein within 1-3 days. BSA loaded silk fibroin hydrogels made withP188 as a gelling excipient reached complete release within 1 day. 4hours into the experiment, cumulative release percentages ranged fromapproximately 66% to approximately 96%. The ratios of silk fibroin toBSA ranged from 2 to 6. Silk fibroin molecular weight or concentration,in the ranges tested, did not affect release kinetics of BSA in thehydrogel formulations with P188. The BSA control sample showed noreduction in concentration over the course of the study. In vitrorelease data for hydrogels prepared with glycerol showed that hydrogelsmade with 120 mb silk fibroin had a higher burst release and reached100% release more quickly than 480 mb silk fibroin hydrogels. 480 mbsilk fibroin hydrogels release approximately 65-80% of BSA by day 1, butthe release then plateaus at day 2. Control BSA solution showedstability over the 2 days of release testing. This relationship betweensilk fibroin molecular weight and release of protein could represent asize dependent release mechanism. Protein release was diffusion based.Since there is minimal hydrolysis and no added enzymes, little to nodegradation of the silk fibroin matrix occurs in vitro. Therefore,decreased release kinetics might be due to a tighter hydrogel networkimpeding the release of BSA. This effect was not observed with the P188formulations. The hydrogel network might be different with the differentgelling agents.

IgG release kinetics from silk fibroin hydrogel formulations withglycerol varied between 38.1% to 59.1% over two days, withoutsignificant release following measured cumulative API release at 2hours. Hydrogels made with 5% silk fibroin (samples 193-03 and 193-04)released more protein by 2 days than those made with 15% silk fibroin(samples 193-07 and 193-08) regardless of the boiling time and molecularweight of the silk fibroin. This result indicated that the silk fibrointo IgG ratio could play a role in diffusion of protein from the silkfibroin formulation. Hydrogels prepared with 5% silk fibroin had a silkfibroin to IgG ratio of 2, while hydrogels prepared with 15% silkfibroin had a silk fibroin to IgG ratio of 6. Hydrogel formulationsprepared with P188 demonstrated lower bursts and released less IgG(maximum release was 45.3%) than those made with glycerol (maximumrelease 59.1%). In general, by two days hydrogels made with 480 mb silkfibroin released more IgG than those made with 120 mb silk fibroin.Interestingly, 15% silk fibroin hydrogels made with P188 released moreIgG than the corresponding hydrogels made with 5% silk fibroin, whichwas the opposite trend observed for the glycerol gels. A hazyprecipitate also formed during formulation of the hydrogels with P188.

Bevacizumab release kinetics from silk fibroin hydrogel formulations allhad similar characteristics. Hydrogels prepared with 5% silk fibroin hada silk fibroin to bevacizumab ratio of 2, while hydrogels prepared with15% silk fibroin had a silk fibroin to bevacizumab ratio of 6. There wasan initial burst phase, followed by a plateau. The burst release varieddependent upon molecular weight of the silk fibroin. 480 mb silk fibroinformulations showed lower initial bursts (measured at 4 hours) ofapproximately 40% while 120 mb silk fibroin formulations showed initialbursts (measured at 4 hours) of 46.4%. The difference increased at 1 dayof release. The formulations with 480 mb silk fibroin (201-1 and 201-2)had released approximately 45% of the protein, while the formulationwith 120 mb silk fibroin (201-3) had released 72.5% of its bevacizumab.The lower burst and lack of release with the 480 mb silk fibroinformulations could be due to a tighter silk network that formed withshorter silk fibroin proteins compared to the larger 120 mb silk fibroinhydrogels. There was no difference in release kinetics betweenformulations with 480 mb silk fibroin concentrations between 5 and 15%.The bevacizumab control displayed that the protein was stable in releasemedia at 37° C. with only a 10% loss maintained over 7 days.

In general, silk fibroin hydrogels showed higher burst and fasterrelease kinetics than the corresponding rod formulations. When comparedto BSA, bevacizumab, and IgG hydrogel formulations, lysozyme (14.7 kDa)released faster than the much larger bevacizumab and IgG molecules(approximately 160 kDa) but more slowly than BSA. Bevacizumab loadedhydrogels containing glycerol released similar levels of protein(45%-70%) as IgG loaded hydrogels with glycerol. Both IgG andbevacizumab loaded hydrogels showed decreased release rate withincreasing silk fibroin concentration. Given the similar size of theseproteins (both approximately 150 kDa), it was possible that the releasewas controlled by diffusion through the silk fibroin network. BSA (66.5kDa) and lysozyme (14.7 kDa) hydrogels released 100% of the protein byday 2, which suggested that smaller proteins diffused more quicklythrough the silk fibroin hydrogel network.

Example 30. Rheological Properties of Silk Fibroin Hydrogels withCelecoxib

The rheological properties of hydrogels loaded with celecoxib (CXB) werestudied. The formulations were prepared as described for the cumulativerelease studies of celecoxib from silk fibroin hydrogels, seen in Table53. To study the rheology, 600 μL of each hydrogel sample was loadedonto the Peltier plate of a Bholin CVOR 150 rheometer. Samples wereanalyzed at 25° C. using a 20 mm parallel plate and a gap of 1.0 mm.Oscillation parameters were set at a frequency of 1 Hz and 0.01% strain.Viscosity was measured at a shear rate of 11/s for 135 seconds, as seenin Table 53. Samples in Table 53 were named by the process used toprepare and formulate each hydrogel. For example, in the sample named120 mb; hyd; 27.8% cxbst; 5% SFf; 10% CXBf; 40% PEG4kf, “120 mb” refersto silk degummed with a 120-minute boil, “hyd” refers to the formulationof the sample as a hydrogel, “27.8% cxbst” refers to a preparation froma stock solution of 27.8% of celecoxib, “5% SFf” refers to a formulationwith 5% (w/v) silk fibroin, “10% CXBf” refers to a formulation with 10%(w/v) celecoxib, and “40% PEG4kf” refers to a formulation with 40% PEG 4kDa. Some hydrogels were prepared with P188 (% P188f).

TABLE 53 Rheology data for hydrogel formulations with celecoxib. Std.Dev. refers to standard deviation. Phase Phase Angle G′ G″ SampleViscosity Viscosity Angle Std. G′ Std. G″ Std. No. Sample name (Pas)Std. Dev. (°) Dev. (Pa) Dev. (Pa) Dev. 168-1 120 mb; hyd; 964.19 182.5510.80 0.54 31982 1516 6086 74 27.8% cxbst; 5% SFf; 10% CXBf; 40% PEG4kf168-2 120 mb; hyd; 324.48 50.86 9.86 1.65 7668 678 1316 82 27.8% cxbst;3% SFf; 10% CXBf; 40% PEG4kf 168-3 120 mb; hyd; 484.94 13.86 8.32 2.7230246 2656 4328 810 27.8% cxbst; 5% SFf; 10% CXBf; 10% P188f 168-4 120mb; hyd; 76.44 5.60 5.35 0.42 4487 274 419 22 27.8% cxbst; 3% SFf; 10%CXBf; 10% P188f 168-5 480 mb; hyd; 238.18 68.89 9.98 2.20 3545 497 60957 27.8% cxbst; 5% SFf; 10% CXBf; 40% PEG4kf 168-6 480 mb; hyd; 43.5514.96 11.79 1.54 503 67 103 3 27.8% cxbst; 3% SFf; 10% CXBf; 40% PEG4kf168-7 480 mb; hyd; 307.25 15.35 8.75 0.28 30825 1609 4737 153 27.8%cxbst; 5% SFf; 10% CXBf; 10% P188f 168-8 480 mb; hyd; 113.16 9.29 8.680.93 9118 667 1385 94 27.8% cxbst; 3% SFf; 10% CXBf; 10% P188f 480 mb;hyd; 27.8% cxbst; 168-9 2% SFf; 59.72 5.47 8.14 0.53 3353 203 478 7 10%CXBf; 10% P188f

The viscosity of the silk fibroin hydrogels was directly related to boththe concentration of silk fibroin and the molecular weight of the silkfibroin in the hydrogel. Higher concentrations of silk fibroin and/orthe use of silk fibroin with a higher average molecular weight yieldedhigher viscosities in otherwise identical formulations. In formulationswith 120 mb silk fibroin, the viscosity was lower for formulations withP188 instead of PEG 4 kDa. For formulations with 480 mb silk fibroin,the viscosity was higher for formulations with P188 instead of PEG 4kDa. Formulations with P188 also had a smaller phase angle than thecorresponding formulation with PEG 4 kDa. The concentration of silkfibroin in a hydrogel demonstrated a direct relationship with thestiffness of the hydrogel, as evidenced by the measured by the storagemodulus (G′) and the loss modulus (G″). Both the G′ and G″ valuesincreased with increasing concentrations of silk fibroin.

Example 31. Injectability of Silk Fibroin Hydrogels with Celecoxib

The formulations were prepared as described for the cumulative releasestudies of celecoxib from silk fibroin hydrogels, seen in Table 54. Theforce required to extrude the hydrogels (injection force) was measured.Each hydrogel sample was mixed back and forth between two syringes toensure homogeneity before being loaded into 1 mL syringe and capped with27G, ½″ needles. The syringes were inserted into a Mark-10 syringecompression fixture and the test stand was set to move the head downonto the syringe plunger and extrude the hydrogel at a rate of 0.5in/min. This was estimated to be equivalent to 0.2 mL/min with thissyringe configuration. The force gauge measured the force required toextrude the hydrogel with a maximum force set at 200 N. Data wascollected over 60 seconds (20 points per second) and exported andgraphed to find where the injectability force plateaued. The averagevalue was taken over this plateau region. Each sample was injected induplicate and average injection force measurements were calculated.

TABLE 54 Analysis of the injectability of silk fibroin hydrogelformulations with celecoxib Injection force (N) at 0.2 mL/minute SampleAverage Average Overall Standard No. Sample name 1 2 Average Deviation168-1 120 mb; hyd; 27.8% cxbst; 5% SFf; 43.5 43.2 43.4 0.2 10% CXBf; 40%PEG4kf 168-2 120 mb; hyd; 27.8% cxbst; 3% SFf; 19.6 20.4 20.0 0.6 10%CXBf; 40% PEG4kf 168-3 120 mb; hyd; 27.8% cxbst; 5% SFf; 16.5 15.2 15.90.9 10% CXBf; 10% P188f 168-4 120 mb; hyd; 27.8% cxbst; 3% SFf; 7.3 8.98.1 1.2 10% CXBf; 10% P188f 168-5 480 mb; hyd; 27.8% cxbst; 5% SFf; 21.321.9 21.6 0.4 10% CXBf; 40% PEG4kf 168-6 480 mb; hyd; 27.8% cxbst; 3%SFf; 9.6 9.6 9.6 0.0 10% CXBf; 40% PEG4kf 168-7 480 mb; hyd; 27.8%cxbst; 5% SFf; 16.1 16.9 16.5 0.5 10% CXBf; 10% P188f 168-8 480 mb; hyd;27.8% cxbst; 3% SFf; 9.4 10.3 9.9 0.7 10% CXBf; 10% P188f 168-9 480 mb;hyd; 27.8% cxbst; 2% SFf; 6.0 6.4 6.2 0.3 10% CXBf; 10% P188f

The experimental results demonstrated a direct relationship between theconcentration of silk fibroin in a hydrogel and the injection force thesilk fibroin hydrogel required. Hydrogels with a higher concentration ofsilk fibroin (e.g. sample 168-1) required a larger injection force toextrude the hydrogel than the corresponding formulation with a lowerconcentration of silk fibroin (e.g. 168-2). In general, the hydrogelsprepared with PEG 4 kDa required higher injection forces than thecorresponding hydrogel with P188. In addition, the molecular weight ofsilk fibroin in the hydrogel was directly related to the injection forcein the hydrogels prepared with PEG 4 kDa. The PEG 4 kDa hydrogelsprepared from higher molecular weight silk fibroin (120 mb) demonstrateda higher injection force than the corresponding hydrogels prepared fromcomparatively lower molecular weight silk fibroin (480 mb).

Example 32. Effect of Select Excipients on Physical Properties ofHydrogels

The injectability experiment as described above was repeated to evaluatethe effect of different excipients on injectability. Silk fibroin wasdegummed as described above, with a 120 mb. Glycerol was purchased fromFisher Chemical (Waltham, Mass.). Celecoxib (CXB) was purchased fromCipla, Miami Fla. Polysorbate-80 was purchased from Croda (Snaith UK).Potassium phosphate monobasic and potassium phosphate dibasic werepurchased from Sigma Aldrich Fine Chemical (SAFC, St. Louis Mo.).

Preparation of Silk Fibroin Hydrogels

To prepare the hydrogels with glycerol, 300 mg of the 120 mb silkfibroin was dissolved in a 20% w/v stock suspension of dry heat treated(DHT) CXB with polysorbate-80 and phosphate buffer to prepare a silk/CXBsuspension with either 7.1% (w/v) or 8.8% (w/v) silk fibroin. Thesuspensions with higher concentration of silk fibroin were used togenerate the hydrogels with higher concentrations of silk fibroin. 2.835mL of the resulting silk/CXB suspension was added to a 6 mL syringe. Thesilk/CXB suspension was then mixed with a second syringe containing2.165 mL of a 92.4% w/v stock solution of glycerol via a B Braun fluiddispensing connector, back and forth until homogeneous (at least 25times). The resulting mixture was then capped with a sterile syringe capand incubated on a rotator overnight at 37° C. The syringes were storedat 4° C. until use.

To prepare the hydrogels with PEG400, 300 mg of the 120 mb silk fibroinwas dissolved in a 20% w/v stock suspension of dry heat treated (DHT)CXB with polysorbate-80 and phosphate buffer to prepare a silk/CXBsuspension with either 7.1% (w/v) or 8.8% (w/v) silk fibroin. Thesuspensions with higher concentration of silk fibroin were used togenerate the hydrogels with higher concentrations of silk fibroin. 2.835mL of the resulting silk/CXB suspension was added to a 6 mL syringe. Thesilk/CXB suspension was then mixed with a second syringe containing2.165 mL of a 92.4% w/v stock solution of PEG400 via a B Braun fluiddispensing connector, back and forth until homogeneous (at least 25times). The resulting mixture was then capped with a sterile syringe capand incubated on a rotator overnight at 37° C. The syringes were storedat 4° C. until use.

The formulations were prepared as described in Table 55. Theformulations tested were named by the method in which they wereprepared. For example, in the sample named “120 mb; hyd; 20% cxbst; 4%SFf; 10% CXBf; 40% Glycf”. “120 mb” refers to silk degummed with a120-minute boil, “hyd” refers to the formulation of the sample as ahydrogel, “20% cxbst” refers to a preparation from a stock solution of20% of celecoxib, “4% SFf” refers to a formulation with 4% (w/v) silkfibroin, “10% CXBf” refers to a formulation with 10% (w/v) celecoxib,and “40% Glycf” refers to a formulation with 40% glycerol. PEG400 wasdenoted in the hydrogels with “PEG400f”.

TABLE 55 Silk fibroin hydrogels with PEG400 or glycerol as excipientsSample % Silk % No. Fibroin Excipient Excipient Sample Name 158-1 4Glycerol 40 120 mb; hyd; 20% cxbst; 4% SFf; 10% CXBf; 40% Glycf 158-2 4PEG400 40 120 mb; hyd; 20% cxbst; 4% SFf; 10% CXBf; 40% PEG400f 158-3 5Glycerol 40 120 mb; hyd; 20% cxbst; 5% SFf; 10% CXBf; 40% Glycf 158-4 5PEG400 40 120 mb; hyd; 20% cxbst; 5% SFf; 10% CXBf; 40% PEG400fInjectability of Silk Fibroin Hydrogels with Select Excipients

The hydrogel samples were loaded into 1 mL syringes. The syringe wascapped with a 27-gauge needle and loaded onto a Mark-10 syringecompression fixture. The test stand was set to extrude the hydrogel at arate of 0.5 inches per minute, which was estimated to be equivalent to0.2 mL/min. The force gauge then measured the force required to extrudethe hydrogel at that rate, with a maximum force set at 200 N. Theinjection forces required to extrude the hydrogel at this rate weremeasured over 60 seconds, with 20 points per second. The data was thenexported and graphed to find where the injectability plateaus. Theaverage value was taken over this range. The results were presented inTable 56. The data showed that using PEG400 as an excipient led toapproximately 25% greater resistance for injection than glycerol. Thehydrogels with glycerol had lower injection forces than thecorresponding hydrogel with PEG400 at all concentrations tested. It wasalso observed that hydrogels with 5% silk fibroin required higherinjection forces than hydrogels with 4% silk fibroin, which wasconsistent with previous observations. All of the hydrogels created werewithin the acceptable injectability range.

TABLE 56 Injectability measurements with different excipients Replicate1 Replicate 2 Overall Sample % Silk % Average Standard Average StandardAverage Standard No. Fibroin Excipient Excipient Force (N) Dev. Force(N) Dev. Force (N) Dev. 158-1 4 Glycerol 40 7.95 0.17 8.12 0.26 8.030.12 158-2 4 PEG400 40 9.85 0.12 10.53 0.15 10.19 0.48 158-3 5 Glycerol40 14.57 0.23 14.59 0.28 14.58 0.01 158-4 5 PEG400 40 18.97 0.34 18.510.13 18.74 0.33Rheology of Silk Fibroin Hydrogels with Select Excipients

The hydrogel samples were loaded onto a Peltier plate system that keptthe temperature at 25° C. The geometry used was a 20 mm parallel plate.The gap was set at 1 mm and the frequency at 1 Hz. Viscosity was takenduring a time sweep at I 1/s over 135 seconds. The experimental resultswere presented in Table 57. In hydrogels having the same silk fibroinconcentration, using glycerol as an excipient created more viscoushydrogels than using PEG400. The effect was more prominent in hydrogelswith 4% silk fibroin than 5%. The glycerol samples were generallystiffer than the PEG400 hydrogels at these two silk fibroinconcentrations as measured by viscosity. However, the glycerol hydrogelsalso had lower injection forces at both concentrations. This differenceindicated that either the glycerol has a positive effect oninjectability, or PEG400 has a negative effect, or some combinationthereof. The glycerol hydrogels could also exhibit more pronouncedshear-thinning behavior than PEG400 hydrogels. This would account forthe lower injection force when under greater shear stress. The moreviscous samples were more likely to be the most cohesive hydrogels invivo.

TABLE 57 Viscosity measurements with different excipients Replicate 1Replicate 2 Average Average Overall Sample % Silk % Viscosity StandardViscosity Standard Average Standard No. Fibroin Excipient Excipient(Pa*s) Dev. (Pa*s) Dev. Force (N) Dev. 158-1 4 Glycerol 40 103.97 2.43138.58 158-1 4 Glycerol 158-2 4 PEG 400 40 62.71 4.56 60.47 158-2 4 PEG400 158-3 5 Glycerol 40 231.07 15.85 281.12 158-3 5 Glycerol 158-4 5 PEG400 40 207.57 11.17 219.53 158-4 5 PEG 400

1. A silk-based product (SBP) comprising processed silk and an oculartherapeutic agent wherein the SBP comprises from about 0.1% to about 98%(w/w) of the ocular therapeutic agent.
 2. The SBP of claim 1, whereinthe SBP is in the shape of a rod. 3-6. (canceled)
 7. The SBP of claim 1,wherein the SBP comprises a ratio of ocular therapeutic agentconcentration to processed silk concentration of from about 0.01 toabout 4.2. 8-9. (canceled)
 10. The SBP of claim 1 comprising at leastone excipient, wherein the at least one excipient is present at aconcentration of from about 0.01% (w/w) to about 20% (w/w).
 11. The SBPof claim 10, wherein the at least one excipient selected from the groupconsisting of lactose, sorbitol, sucrose, mannitol, lactose USP, Starch1500, microcrystalline cellulose, Avicel, phosphate salts, sodiumchloride, hydrochloric acid, polysorbate 80, potassium phosphatemonobasic, potassium phosphate dibasic, sodium phosphate dibasic, sodiumphosphate monobasic, phosphate buffer, phosphate buffered saline, sodiumhydroxide, dibasic calcium phosphate dehydrate, tartaric acid, citricacid, fumaric acid, succinic acid, malic acid, polyvinylpyrrolidone,copolymers of vinylpyrrolidone and vinylacetate, hydroxypropylcellulose,hydroxyethylcellulose, hydroxypropylmethylcellulose, polyvinyl alcohol,polyethylene glycol, acacia, and sodium carboxymethylcellulose.
 12. TheSBP of claim 11, wherein one of the at least one excipient is sucrose.13-14. (canceled)
 15. The SBP of claim 1, wherein the SBP is in theshape of a rod and wherein the rod has a diameter of from about 0.1 mmto about 1.5 mm.
 16. (canceled)
 17. The SBP of claim 1, wherein the SBPcomprises a hydrogel.
 18. (canceled)
 19. The SBP of claim 17, whereinthe SBP comprises at least one excipient, and wherein the at least oneexcipient is selected from the group consisting of sorbitol,triethylamine, 2-pyrrolidone, alpha-cyclodextrin, benzyl alcohol,beta-cyclodextrin, dimethyl sulfoxide, dimethylacetamide (DMA),dimethylformamide, ethanol, gamma-cyclodextrin, glycerol, glycerolformal, hydroxypropyl beta-cyclodextrin, kolliphor 124, kolliphor 181,kolliphor 188, kolliphor 407, kolliphor EL (cremaphor EL), cremaphor RH40, cremophor RH 60, dalpha-tocopherol, PEG 1000 succinate, polysorbate20, polysorbate 80, solutol HS 15, sorbitan monooleate, poloxamer-407,poloxamer-188, Labrafil M-1944CS, Labrafil M-2125CS, Labrasol, Gellucire44/14, Softigen 767, mono- and di-fatty acid esters of PEG 300, PEG 400,or PEG 1750, kolliphor RH60, N-methyl-2-pyrrolidone, castor oil, cornoil, cottonseed oil, olive oil, peanut oil, peppermint oil, saffloweroil, sesame oil, soybean oil, hydrogenated vegetable oils, hydrogenatedsoybean oil, medium chain triglycerides of coconut oil, medium chaintriglycerides of palm seed oil, beeswax, d-alpha-tocopherol, oleic acid,medium-chain mono-glycerides, medium-chain di-glycerides,alpha-cyclodextrin, betacyclodextrin, hydroxypropyl-beta-cyclodextrin,sulfo-butylether-beta-cyclodextrin, hydrogenated soyphosphatidylcholine, distearoylphosphatidylglycerol,L-alphadimyristoylphosphatidylcholine,L-alpha-dimyristoylphosphatidylglycerol, PEG 300, PEG 300caprylic/capric glycerides (Softigen 767), PEG 300 linoleic glycerides(Labrafil M-2125CS), PEG 300 oleic glycerides (Labrafil M-1944CS), PEG400, PEG 400 caprylic/capric glycerides (Labrasol), polyoxyl 40 stearate(PEG 1750 monosterate), polyoxyl 8 stearate (PEG 400 monosterate),polyvinyl pyrrolidone, propylene carbonate, propylene glycol, solutolHS15, sorbitan monooleate (Span 20), sulfobutylether-beta-cyclodextrin,transcutol, triacetin, 1-dodecylazacyclo-heptan-2-one, caprolactam,castor oil, cottonseed oil, ethyl acetate, medium chain triglycerides,methyl acetate, oleic acid, safflower oil, sesame oil, soybean oil,tetrahydrofuran, glycerin, and PEG 4 kDa.
 20. The SBP of claim 19,wherein the at least one excipient is poloxamer-188.
 21. The SBP claim19, wherein the at least one excipient is PEG 4 kDa.
 22. The SBP claim19, wherein the at least one excipient is glycerol.
 23. The SBP claim 1,wherein the processed silk comprises silk fibroin, and wherein the silkfibroin is present in the SBP at a concentration of from about 0.1%(w/v) to about 30% (w/v). 24-25. (canceled)
 26. The SBP claim 1, whereinthe SBP has an osmolarity which is from about 275 mOsm to about 285mOsm. 27-32. (canceled)
 33. The SBP of claim 1, wherein the oculartherapeutic agent comprises a non-steroidal anti-inflammatory drug(NSAID).
 34. The SBP of claim 33, wherein the NSAID comprises one ormore of aspirin, carprofen, celecoxib, deracoxib, diclofenac,diflunisal, etodolac, fenoprofen, firocoxib, flurbirofen, ibuprofen,indomethacin, ketoprofen, ketorolac, mefenamic acid, meloxicam,nabumetone, naproxen, oxaprozin, piroxicam, robenacoxib, salsalate,sulindac, and tolmetin.
 35. The SBP of claim 34, wherein the NSAID iscelecoxib. 36-37. (canceled)
 38. A pharmaceutical composition, whereinthe pharmaceutical composition comprises the SBP of claim 1 and at leastone excipient, wherein the SBP is formulated for intraocularadministration.
 39. The pharmaceutical composition of claim 38, whereinthe SBP is formulated for one or more of intravitreal administration,intraretinal administration, intracorneal administration, intrascleraladministration, punctal administration, administration to the anteriorsub-Tenon's, suprachoroidal administration, administration to theposterior sub-Tenon's, subretinal administration, administration to thefornix, administration to the lens, and intra-aqueous humoradministration. 40-45. (canceled)
 46. A method of treating a subjectcomprising contacting the subject with the pharmaceutical composition ofclaim
 38. 47. The method of claim 46, wherein the subject comprises anocular indication.
 48. (canceled)
 49. The method of claim 47, whereinthe ocular indication comprises one or more of an infection, refractiveerrors, age related macular degeneration, cystoid macular edema,cataracts, diabetic retinopathy (proliferative and non-proliferative),glaucoma, amblyopia, strabismus, color blindness, cytomegalovirusretinitis, keratoconus, diabetic macular edema (proliferative andnon-proliferative), low vision, ocular hypertension, retinal detachment,eyelid twitching, inflammation, uveitis, bulging eyes, dry eye disease,floaters, xerophthalmia, diplopia, Graves' disease, night blindness, eyestrain, red eyes, nystagmus, presbyopia, excess tearing, retinaldisorder, conjunctivitis, cancer, corneal ulcer, corneal abrasion, snowblindness, scleritis, keratitis, Thygeson's superficial punctatekeratopathy, corneal neovascularization, Fuch's dystrophy,keratoconjunctivitis sicca, iritis, chorioretinal inflammation (e.g.chorioretinitis, choroiditis, retinitis, retinochoroiditis, parsplanitis, Harada's disease, aniridia, macular scars, solar retinopathy,choroidal degeneration, choroidal dystrophy, choroideremia, gyrateatrophy, choroidal hemorrhage, choroidal detachment, retinoschisis,hypertensive retinopathy, Bull's eye maculopathy, epiretinal membrane,peripheral retinal degeneration, hereditary retinal dystrophy, retinitispigmentosa, retinal hemorrhage, retinal vein occlusion, and separationof retinal layers. 50-51. (canceled)
 52. The method of claim 47, whereinthe pharmaceutical composition is administered to the subject viaintravitreal administration. 53-57. (canceled)
 58. The method of claim46, wherein contacting the subject with the pharmaceutical compositionresults in a concentration of the ocular therapeutic agent in an eye ofthe subject of from about 0.01 ng/mL to about 60,500 ng/mL. 59-66.(canceled)
 67. The method of claim 58, wherein the ocular therapeuticagent is detectable in one or more components of the eye for at least 3months. 68-77. (canceled)
 78. The method of claim 67, wherein the eyecomponent is selected from the group consisting of the retina, thevitreous humor, and/or choroid. 79-105. (canceled)